Proteins with predictable liquid-liquid phase separation

Abstract

Providing a protein with predictable liquid-to-liquid phase separation. [Solution] Peptide biomacromolecules exhibiting controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration are described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 985,179, filed on 4 March 2020, which is incorporated herein by reference in its entirety. Research funded by the federal government This invention was made with the support of the U.S. Government under National Institutes of Health Grant No. R35GM127042 and National Science Foundation Grant No. DMR-17-29671. The U.S. Government has certain rights in this invention. Sequence List This application is filed together with a computer-readable sequence listing in accordance with 37C.FR §1.821(c). A text file was submitted by EFS, “028193-9340-WO01_sequence_listing_2-MAR-2021_ST25.txt”, created on March 2, 2021, containing 317 sequences, having a file size of 527 kilobytes, and is incorporated herein by reference in its entirety. Technical field Peptide biomacromolecules exhibiting controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration are described herein. [Background technology]

[0002] Essentially disordered proteins (IDPs) are well recognized for their roles in various biological (dysfunctional) functions. A subset of IDPs, called biological condensates, physically separate from the cytoplasm and control the accessibility of various macromolecules. While our ability to detect protein disorder has advanced rapidly thanks to sophisticated statistical methods, our ability to predict phase separation has lagged significantly. Predicting phase separation is not regular because numerous variables influence it. Roughly speaking, these include (1) the amino acid composition and pattern of the primary protein sequence; (2) heterotype interactions with RNA or other macromolecules; and (3) the quality of the solvent. Although many studies have described attempts to predict IDP phase behavior, few have directly addressed this problem. Given its recognized importance to cellular function, now is the time for active research, and much effort is underway using computational and experimental approaches. However, to date, most experimental methods for developing sequence-level understanding of IDP phase behavior rely on mutation strategies of native IDPs with coarse residue-level or domain-level mutations. What is needed are peptide biomacromolecules containing essentially disordered proteins that exhibit controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration. [Overview of the Initiative]

[0003] One embodiment described herein is (X-Z1-X-Z2-Z3-X-Z4-Z3) n(In the formula, X is proline (P) or glycine (G), the P:G ratio is any number, Z1 is arginine (R), aspartic acid (D), or lysine (K), the R:D ratio is any number, and the K:R ratio can be any number, Z2 is Asp(D), Arg(R), or Glu(E), the R:D ratio can be any number, and the D:E ratio can be any number, Z3 is asparagine (N), glutamine (Q), serine (S), or s The polypeptide has controlled reversible phase separation and comprises 10 or more repeat sequences of amino acid sequences, where Z4 is rheonine (T), and the ratio between N:Q:S:T can be any number; and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number. In one embodiment, X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. In another embodiment, Z1 is arginine (R), aspartic acid (D), or lysine (K), and the R:D ratio does not exceed 1:5, and the K:R ratio can be any number. In another embodiment, phase separation depends on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration. In another embodiment, n is 10 to 200. In another embodiment, the molecular weight is at least 5 kDa to 500 kDa. In another embodiment, the molecular weight is about 5 kDa to about 100 kDa. In another embodiment, the phase separation temperature is 0 to 100°C. In another embodiment, the phase separation temperature is 4 to 25°C, about 25°C, 25 to 37°C, about 37°C, 35 to 38°C, or >38°C. In another embodiment, the polypeptide comprises modified amino acids, a reporter protein, or an enzyme. In another embodiment, the sequence is (GRGDSPYS) m(wherein m is 20 to 80) includes. In another embodiment, polypeptide includes sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, Includes an array selected from 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279, or one or more combinations thereof.

[0004] Another embodiment described herein is (X-Z1-X-Z2-Z3-X-Z4-Z3) n(In the formula, X is proline (P) or glycine (G), the P:G ratio is any number, Z1 is arginine (R), aspartic acid (D), or lysine (K), the R:D ratio is any number, the K:R ratio can be any number, Z2 is Asp(D), Arg(R), or Glu(E), the R:D ratio can be any number, the D:E ratio can be any number, and Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) A pharmaceutically acceptable composition comprising a polypeptide having controlled reversible phase separation comprising 10 or more repeat sequences of amino acid sequences (where the ratio between N:Q:S:T can be any number, and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number). In one embodiment, X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. In another embodiment, Z1 is arginine (R), aspartic acid (D), or lysine (K), and the R:D ratio does not exceed 1:5, and the K:R ratio can be any number. In another embodiment, the composition includes antimicrobial peptides selected from the following: antibody-binding domains derived from Staphylococcus protein A(ZD) (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelicidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein The conjugation molecule further includes one or more of the following: phosphate drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV-binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195).In another embodiment, the composition enhances the bioavailability of the bound molecule compared to the bound molecule in its free form. In another embodiment, the composition enhances the expression of the bound molecule compared to the bound molecule in its free form. In another embodiment, the composition enhances the stability of the bound molecule compared to the bound molecule in its free form. In another embodiment, the composition enhances the stability of the bound molecule during expression in prokaryotes and eukaryotes compared to the bound molecule in its free form. In another embodiment, the enhanced stability includes resistance to denaturation during freezing, thawing, or lyophilization. In another embodiment, the composition modulates enzymatic, metabolic, or physiological function within a cell or organism. In another embodiment, the modulation reduces the bioavailability of the bound molecule. In another embodiment, the bound molecule comprises a therapeutic or cytotoxic protein or peptide.

[0005] Another embodiment described herein is a method for enhancing the bioavailability or stability of a protein, the method comprising one or more proteins and (X-Z1-X-Z2-Z3-X-Z4-Z3) n(In the formula, X is proline (P) or glycine (G), the P:G ratio is any number, Z1 is arginine (R), aspartic acid (D), or lysine (K), the R:D ratio is any number, the K:R ratio can be any number, Z2 is Asp(D), Arg(R), or Glu(E), the R:D ratio can be any number, the D:E ratio can be any number, and Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T)) The method involves creating a polypeptide fusion protein having controlled reversible phase separation, comprising 10 or more repeat sequences of amino acid sequences, where the ratio between N:Q:S:T can be any number, and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number. In one embodiment, X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. In another embodiment, Z1 is arginine (R), aspartic acid (D), or lysine (K), and the R:D ratio does not exceed 1:5, and the K:R ratio can be any number. In one embodiment, X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. In another embodiment, Z1 is arginine (R), aspartic acid (D), or lysine (K), and the R:D ratio is not greater than 1:5, and the K:R ratio can be any number.In another embodiment, the protein is selected from the antibody-binding domain (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), cathelicidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), and H5-61-90 (SEQ ID NO: 175) derived from Staphylococcus protein A(ZD). Antimicrobial peptides; RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domain (Yifan or FMRP (SEQ ID NO: 39)); 191)); or comprising one or more AAV-conjugated peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195). In another embodiment, the enhanced bioavailability of the fusion protein can be used for the isolation or separation of biological molecules. In another embodiment, the biological molecule comprises one or more lipids, cells, proteins, nucleic acids, carbohydrates, or viral particles. In another embodiment, the nucleic acid is single-stranded or double-stranded DNA or RNA. In another embodiment, the viral particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measles virus particle, or a herpesvirus particle. In another embodiment, the protein comprises albumin, a monoclonal IgG antibody, or an Fc fusion antibody. In another embodiment, isolation or separation is achieved via reversible phase separation. This patent or application includes at least one color drawing. A copy of this patent or patent application publication containing the color drawing(s) will be provided by the Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0006] [Figure 1A-G]This paper demonstrates that artificial intrinsically disordered polypeptides (A-IDPs), inspired by native IDPs, exhibit reversible UCST phase behavior. Figure 1A shows that proteomic analysis of native IDPs forming biomolecular condensates reveals they possess high G / P, charged and uncharged polar residues, yet still exhibit a total charge equilibrium. Figure 1B shows an example of a high-density exclusionary phase formed by UCST-exhibiting A-IDPs, even in a complex medium of bacterial cell lysates. Coacervates were observed to achieve almost complete separation from all other cellular proteins and debris present in the cell lysates after centrifugation, demonstrating that this facilitates purification from insoluble cell lysate fractions without the use of affinity tags. Figure 1C shows an example of an SDS-PAGE gel of a set of A-IDPs with conserved sequences but gradually increasing MW, [Q5,8]-20 to [Q5,8]-80, exhibiting high purity A-IDPs obtained by utilizing their UCST phase behavior without requiring any chromatographic purification. Figure 1D shows visualization of UCST phase separation of [Q5,8]-20 in a water-in-oil droplet using a fluorescence microscope. During cooling, phase separation in the droplet begins at multiple sites, and the spots growing from each site slowly merge into a single compact phase. During reheating, the equilibrium state with the surrounding dilute phase is always re-established, leading to a smaller volume occupied by a higher concentration of dilute and compact phases. φ=0.0018, scale bar=50μm. Figure 1E shows a schematic UCST phase diagram of the cooling and heating cycle of UCST polypeptides in a water-in-oil droplet. Figure 1F shows dynamic light scattering data of [Q5,8]-20, illustrating the change in hydrodynamic radius during cooling. Upon reaching the cloud point, [Q5,8]-20 transitions from a soluble monomer polypeptide with a hydration radius of 5-6 nm to micron-sized aggregates. Data were collected in 140 mM PBS, pH 7.4, with φ = 0.0043. Figure 1G shows that the UCST cloud point is influenced by the volume fraction of polypeptides in the solution. This behavior follows a natural logarithmic dependence in dilute regimes (R² = 0.98). [Figure 2][WT]-20-sfGFP exhibits phase separation memory during multiple heating and cooling cycles. During multiple heating and cooling cycles, [WT]-20-sfGFP forms spots in the same locations as during the first cooling cycle. Given the significance of this memory, it is important to note that the observed transition temperature was below room temperature (approximately 15°C), suggesting that these cells were insensitized to phase separation, having been incubated at 37°C and treated at room temperature. The scale bar indicated 5 μm. The cooling and heating rates were set to a constant 5°C / min. [Figure 3A-B] Additional proteomics analysis. Figure 3A shows a graph of the difference in amino acid composition between ordered and disordered regions within the same protein. Disordered regions are defined as having a score of >0.5 using PONDR VSL2, and ordered regions are defined as having a score of <0.5. The values ​​were calculated by subtracting the percentage of chain composition in the disordered regime from that of the ordered regime. Bars represent the 25th–75th percentiles, and whiskers represent the 10th–90th percentiles. The central line represents the median of the dataset. N=63, *P<0.01 in Student's t-test between ordered and disordered regions of all sampled proteins. Figure 3B shows a histogram plot of the lengths of disordered regions analyzed in this study. Bars represent the 25th–75th percentiles, and whiskers represent the 10th–90th percentiles. [Figure 4] Figures 1-5 relate to the SDS-PAGE gels of the purified proteins used in this study. The lane labeling for each purified protein in this study is listed in Tables 2 and 3. [Figure 5] Wide-field fluorescence microscope image of fluorescently labeled [Q5,8]-20 within the water-in-oil component. [Q5,8]-20 was labeled with AlexaFluor350 via NHS chemistry and resuspended in 140 mM PBS, pH 7.4 to a final φ = 0.003. The water-in-oil mixture was transferred to a glass slide and cooled from 50°C to 10°C. Scale bar = 20 μm. [Figure 6]Additional dynamic light scattering data. Data collected on a 20 nm filtered sample at a volume fraction predicted to show liquid-liquid phase separation at 40°C. Data collected in 140 mM PBS, pH 7.4. [Figure 7] The cooling and heating cycle exhibits minimal hysterical behavior. The phototurbidity measured at 350 nm is observed in the repeated cooling and heating curves of [Q5,8]-20 at φ=0.0025, 40°C to 30°C. [Figure 8A-F] Control of UCST cloud point using the main chain amino acid composition is shown. Figure 8A shows a schematic diagram describing the methodology of mixing repeat sequence unit b with a homopolymer of a. A high UCST cloud point WT A-IDP consisting of 40 repeat sequences of GRGDSPYS, which is a, is mixed with an increasing fraction of repeat sequence b, which is GRGDQPYQ, to investigate the "loss of function" in the UCST phase behavior of the polymer of a. The incorporation of b into a is designed to ensure mixing of the two repeat sequences along the polypeptide chain and minimize uneven behavior. Figure 8B shows the incorporation of mutant IDPs of b into the results, where the UCST cloud point temperature (Tt) of each mutant IDP is a linear function of the volume fraction (φ) of A-IDP. Figure 8C shows that the effect of the composition, i.e., the degree of incorporation, is a linear function of the degree of substitution of b into a at a constant volume fraction of 10⁻³ (R²=0.97). Figure 8D shows that substitution of an aromatic Y residue with an aliphatic V dramatically reduces Tt. Figure 8E shows that substitution of R with K dramatically reduces Tt. A 50% substitution of K to R lowers Tt by more than 40°C. Figure 8F shows that chemical composition can affect saturation concentration by a factor of 100 for a given molecular weight (Csat = 1~800 μm at 37°C). This is preferably about 1 μm and can be visualized by normalizing to the saturation concentration of [WT]-40, indicated by the horizontal dotted line. [Figure 9A-E]This shows the effect of single amino acid substitutions on the UCST cloud point and a novel relative UCST tendency scale. Figure 9A shows the partial binodal phase boundaries of well-mixed diblock polypeptides with various aromatic:aliphatic residue ratios. Figure 9B shows the partial binodal phase boundaries of well-mixed diblock polypeptides with various polarity uncharged residue ratios. Figure 9C shows the partial binodal phase boundaries of well-mixed diblock polypeptides with various identity positive and negative charged residues. Figure 9D shows the partial binodal phase boundaries of well-mixed diblock polypeptides with varying amounts. Data were collected under physiological solution conditions (140 mM PBS, pH 7.4) at φ = 10⁻³. All polypeptides are 326 amino acid long. Figure 8E shows the relative scale of UCST tendency based on substitutions made to the [WT] repeat sequence motif. If the amino acid on the left is substituted with the amino acid on the right of the arrow, and the total number of amino acids is 326, the listed transition temperatures are the UCST cloud points at φ = 10⁻³. [Figure 10] This section shows the analysis of secondary structure using circular dichroism (CD) spectroscopy. CD spectra of various A-IDPs lack the defined secondary structure curve shape features of other IDPs and other repeating protein polymers. Data were collected at 5 mM PBS, pH 7.4, 5 μm, 50°C (soluble chain). Error bars indicate the standard deviation of three consecutive runs. [Figure 11A-C] This shows the control of UCST cloud point by the molecular weight of A-IDP. Figure 11A shows that the molecular weight of the polypeptide affects Tt. Figure 11B shows the direct Tt scale using the natural logarithm of MW. Figure 11C shows that, at a given chemical composition, it is possible to adjust Csat by more than five orders of magnitude simply by changing the MW of A-IDP (Csat = 1 nM ~ 400 μm at 37°C). [WT]-40 has a Csat of approximately 1 μm. [Figure 12A-D]This shows a slight effect on UCST clouding in protein polypeptides. Figure 12A shows a partial binodal phase diagram of sequence syntax rearrangement focused around the Pro residue. It is evident that mutations affect the UCST binodal, particularly the UCST binodal at position 5, but do not eliminate the phase behavior. Data were collected under physiological conditions (140 mM PBS, pH 7.4). Figure 12B shows a partial binodal phase boundary of a non-agnostical but compositionally identical version of [WT]-20. Figure 12C shows the turbidity curves of [H7]-60 in solutions of different pH. Decreasing pH and protonation of the His residue increase and broaden the observed UCST phase behavior. This effect is concentrated at around pH 7, very closely tied to the predicted pKa of the imidazole group at H. In contrast, the UCST clouding of [WT]-60 does not change as a function of pH (black dots, graph inset). Figure 12D shows the turbidity curves of [Q5,8]-40 in solutions containing different concentrations of NaCl. In pure water, [Q5,8]-40 exhibits a broad transition at higher temperatures. Increasing the concentration of NaCl from 0 to 140 mM lowers and sharpens the UCST cloud point, eventually reaching a minimum of approximately 500 mM. From this point, the protein exhibits a salting-out effect, and the transition temperature begins to rise again. [Figure 13A-C]Phase diagram creation using a temperature gradient instrument. Figure 13A shows a typical dark-field image of the [Q5,8]-20 solution on a temperature gradient instrument. The transition temperatures of the reference solution (red and blue lines) and the 20 mg / mL [Q5,8]-20 solution (green line) are indicated by horizontal color lines. The vertical crimson dotted line along the 20 mg / mL capillary tube illustrates the area of ​​the image used to measure the line scan. Figure 13B shows the line scan of normalized light scattering intensity versus temperature for the 20 mg / mL [Q5,8]-20 capillary shown in Figure 13A. The black dotted line represents the tangent to the high temperature baseline and the increase in light scattering at lower temperatures. These two lines intersect at Tph, as indicated by the vertical green line. Figure 13C shows the final binodal phase lines of [WT]-20 and [Q5,8]-20 using multiple data points from the temperature gradient instrument. Using a three-piece fit, three regimes were approximated to roughly correspond to the diluted, overlapping, and semi-diluted regimes of the polypeptide phase diagram. The observed data and subsequent approximations show that polypeptide sequences influence the UCST cloud point not only in the diluted regime but also across the entire measured concentration range (φ≦0.5). [Figure 14A-B] This shows the quantification of dextran incorporation during A-IDP phase separation. Figure 14A shows fluorescence microscopy images of phase-separated droplets in the presence of dextran molecules of different molecular weights (10 / 40 kDa) labeled with Alexa488 (green) fluorophores. Within the phase-separated space (dark circles), there is very slight segregation of dextran molecules as dextran molecular weight or as a function of the A-IDP sequence. The scale bar is 20 μm. Figure 14B shows the quantification of fluorescence signals between the internal and external ranges of the phase-separated droplets. [Figure 15A-G]This shows that A-IDP exhibits regulated intracellular droplet formation based on molecular weight and aromatic:aliphatic content ratio. All scale bars are 5 μm. Figure 15A shows a schematic diagram illustrating the use of two key parameters, aromatic:aliphatic content ratio and molecular weight, to control intracellular droplet formation by modulating Csat. Figure 15B shows a partial in vitro binodal of the A-IDP-sfGFP fusion in a dilute regime in 140 mM PBS, pH 7.4. Similar to A-IDP, the A-IDP-GFP fusion protein exhibits molecular weight and aromatic content-dependent phase behavior. Figure 15C shows phase separation of the [WT]-20-sfGFP fusion in eukaryotic cells (HEK293 cells, day 5). Instead of the formation of a single droplet observed in vitro in primitive cells (see Figure 1C), many distinct droplets are formed, indicating either diffusion-limited or rest-limited coalescence. Figure 15D shows confocal fluorescence images of A-IDP-sfGFP in E. coli as a function of induction time and molecular weight. Higher intracellular concentrations are required for intracellular droplet formation for [WT]-20 versus [WT]-40. It is noteworthy that [WT]-40 has a lower lysis phase of A-IDP with a lower φ' outside the denser droplet phase compared to [WT]-20. Figure 15E shows that a decrease in aromatic content increases Csat in a volume-dependent manner. Figure 15F shows that A-IDP-sfGFP fusions exhibit an order-of-magnitude shift in their Csat, determined by their molecular weight and aromatic:aliphatic content ratio. Figure 15G shows the magnitude of intracellular droplet (φ'' or dense phase) growth with respect to induction time. As the concentration of A-IDP-sfGFP increases intracellularly, the lysis concentration outside the droplet does not change (Figure 19), but the magnitude of intracellular droplet growth changes relative to the total cellular range. The images show individual cells from a [3Y7:V7]-40-sfGFP culture at various time points. Error bars represent the standard error of the mean. [Figure 16A-C]A comparison of the partial binodal phase diagrams of A-IDP and A-IDP-sfGFP fusions is shown. Figure 16A shows the partial binodal phase boundary of [WT]-40 and [WT]-40-sfGFP. Figure 16B shows the partial binodal phase boundary of [3Y7:V7]-40 and [3Y7:V7]-40-sfGFP. Figure 15C shows the partial binodal phase boundary of [WT]-20 and [WT]-20-sfGFP. The sfGFP fusions lower the UCST binodal line for all A-IDPs. These data suggest that the larger the molecular weight of the polypeptide, the less it is affected by the sfGFP fusion, as the observed difference between [WT]-40 and [3Y7:V7]-40 is only about 10°C instead of about 20°C for [WT]-20. [Figure 17] This image shows confocal microscopy images of HEK293 cells transiently transfected with [WT]-20-sfGFP. Confocal fluorescence image slices within the cells show that phase-separated droplets form in the cytoplasm, clearly without coexistence with other cellular structures. The image was acquired 24 hours after transfection with 3 μg of the pCDNA plasmid encoding [WT]-20-sfGFP. Scale bar = 5 μm. [Figure 18] The total cell fluorescence measurements are shown as a function of induction time. E. coli cultures were spun down and resuspended in 140 mM PBS, pH 7.4. sfGFP light turbidity and fluorescence intensity were measured and plotted as a function of time. Data were collected at 22°C. [Figure 19] The images show measurements of cellular fluorescence at different locations within the cell. Digital separation was performed using ImageJ between the dense phase separation range and the soluble cytoplasmic space of the cell. The mean total cellular fluorescence intensity (solid line) and cytoplasmic fluorescence intensity (dotted line) are plotted as a function of time after IPTG induction. [WT]-20-sfGFP does not show intracellular droplets until the 6-hour mark. At this point, the cytoplasmic fluorescence intensity remains constant, but the total fluorescence increases from 6 hours onward. The [WT]-40-sfGFP phase transitions before the 2-hour time point. [Figure 20A-D]Figure 20A shows that A-IDP exhibits reversible coacervation in E. coli, determined by their molecular weight and aromatic:aliphatic ratio. All scale bars are 5 μm. Figure 20A shows that intracellular droplets containing [WT]-20-sfGFP can be formed and reversibly dissolved via alternative cooling and heating cycles. This process is completely reversible over four rounds of cooling and heating. Cooling rate = 5°C / min, induction time 4 hours. Figure 20B shows that the Tt normalized for intracellular fluorescence of sfGFP in each individual cell (n=30) does not change significantly over four heating (red bars) and cooling (blue bars) cycles. Squares indicate the 25th–75th percentile. Figure 20C shows that the intracellular Tt, similar to that in vitro, is a function of A-IDP molecular weight and aromatic content. Cooling gradient = 60°C → 10°C. Cooling rate = 5°C / min, A-IDP gene induction time 8 hours. Whiskers are shown at the 10th–90th percentile. Figure 20D shows intracellular binodal lines of various A-IDP-sfGFP fusions. Tt increases as a function of cellular fluorescence, alternative A-IDP concentration, and aromatic content of A-IDP. Data analyzed at 2, 4, and 8 hours for [WT]-40-sfGFP and [3Y7:V7]-10, and at 4, 8, and 24 hours for [Y7:V7]-40 (n=30). Error bars indicate the standard error of the mean. Figure 20D shows that the solubility of the reconstituted GFP-A-IDP complex can be regulated with temperature during sfGFP reconstitution in the dense phase. Data were collected 36 hours after IPTG induction and 12 hours after arabinose induction. [Figure 21A-C]The image analysis of the number of spots formed in each cell is shown. For each histogram, 100 cells were randomly placed in a table. Figure 21A shows the number of intracellular spots formed in each cell containing [WT]-20-sfGFP during a cooling gradient from 60°C to 10°C (green), and imaged isothermally at 22°C. Isothermal analysis was performed 6 hours after induction, and intracellular spots were observed at the first time point. The cooling gradient was performed 4 hours after induction, and the transition temperature (Tt) was 22°C to 37°C. Figure 21B shows the number of intracellular spots formed in each cell containing [3Y:V]-40-sfGFP during a cooling gradient from 60°C to 10°C (green), and imaged isothermally at 22°C. Isothermal analysis was performed 4 hours after induction, and intracellular spots were observed at the first time point. The cooling gradient was performed 4 hours after induction, and Tt was 22°C to 37°C. Figure 21C shows the number of intracellular spots formed in each cell containing [[WT]-40-sfGFP during a cooling gradient from 60°C to 10°C (green), and imaged isothermally at 22°C. Isothermal analysis was performed 4 hours after induction, and intracellular spots were observed at the first time point. The cooling gradient was performed 4 hours after induction, but the observed transition was >37°C, which suggests the possibility of memory. [Figure 22A-F]This shows manipulated intracellular droplets using programmable functions. Figure 22A shows site-specific labeling of droplets using small molecule fluorescent dyes. E. coli cells containing condensates formed by a [3Y7:V7]-40 variant having an azide-phenylalanine (AzF) residue that presents a double orthogonal azide that can be labeled at insights using a dibenzocyclooctin dye conjugate (DBCO-Alexa488). The DBCO-Alexa488 mixture can be diffused into the cells and into the intracellular A-IDP condensates, and the azide group can be labeled within 10 minutes of incubation with live E. coli. Figure 22B shows the reconstitution of functional GFP in the condensates by recruitment from the partner cytoplasm using separate GFP systems. The GFP-11-[3Y7:V7]-40 fusion protein can recruit GFP-1-10 from the surrounding cytoplasm into the intracellular droplet. During the formation of intracellular condensates 24 hours after IPTG induction of GFP-11-[3Y7:V7]-40 (left panel), subsequent GFP-1-10 induction by arabinose allows for the recruitment of GFP-10 into the condensate and the reconstitution of functional sfGFP within existing intracellular condensates within 12 hours of GFP-1-10 induction (right panel). Figure 22C shows a schematic diagram of the enzyme-condensate experiment. The α-peptide of LacZ (αp) is fused to a fluorescent reporter protein (mRuby3) and expressed from an IPTG-inducible gene derived from a plasmid in the E. coli strain KRX, which has a deletion mutation in the LacZ gene that produces a cleaved, catalytically inactive enzyme lacking αp. The complementarity of LacΔM15 via αp-A-IDP-mRuby3 fusion produces an active enzyme that converts FDG to fluorescein and is then rapidly transported from the intracellular space to the surrounding culture medium. Figure 22D shows a confocal microscope image illustrating the fluorescence conversion of fluorescein di-β-D-galactopyranoside (FDG). Note that the mottled structures of αp-mRuby3 in the upper panel are due to the fractionation of the fusion product, forming inclusion bodies in the cell. When αp fuses with [WT]-20-mRuby3, fluorescence is first observed at the site of the intracellular phase transition in the coacervate droplet, after which fluorescein diffuses into the cytoplasm and then into the extracellular space.The increasing molecular weight of A-IDP leads to FDG conversion at an earlier time point and higher overall conversion after 20 minutes. Re-equilibrium images of αp-[WT]-40-mRuby3 and αp-[WT]-80-mRuby3 can be seen in Figure 25 for improved visualization of the coexistence of intracellular droplets and converted FDG. Figure 22E shows the intracellular concentration of fluorescein produced by catalytic conversion of FDG and standardized against the mRuby3 fluorescence of each individual cell (n≒300). Catalytic efficiency increases with A-IDP MW, as seen by the larger ratio of green fluorescence resulting from FDG conversion to fluorescein, standardized against the red fluorescence of molar-based mRuby3. Both αp-[WT]-40-mRuby3 and αp-[WT]-80-mRuby3 show statistically significant differences from the control (αp-mRuby3). Error bars indicate the standard error of the mean. Figure 22F shows that all αp-A-IDP-mRuby3 fusions exhibit a higher ratio of intracellular green fluorescence, indicating a longer persistence of fluorescent FDG in intracellular space compared to the αp-mRuby3 control. Error bars indicate the mean standard error. All scale bars are 5 μm. [Figure 23A-B] Confocal microscopy images of separate GFP recruitment into intracellular droplets are shown. Figure 23A shows that GFP-11-[3Y7:V7]-40-mRuby3, co-expressed in the presence of GFP-1-10, produces fluorescent GFP only within the droplet. Figure 23B shows the presence of slight green fluorescence within the intracellular droplet in the absence of GFP-1-10 induction. Data acquired at 22°C. Scale bar = 5 μm. [Figure 24] This study demonstrates that A-IDP can regulate the solubility of endogenously bound molecule 2. During recruitment of sfGFP into the dense phase, the solubility of the complete complex can be controlled using temperature. Data were collected 36 hours after IPTG induction and 12 hours after arabinose induction. Scale bar = 5 μm. [Figure 25A-B]Color equilibrated confocal microscopy images of αp-[WT]-40-mRuby3 and αp-[WT]-80-mRuby3 are shown. All scale bars are 5 μm. Figure 25A shows a color re-equilibrium image of Figure 22B for improved visualization of intracellular droplets formed by αp-A-IDP-mRuby3 fusions. Figure 25B shows separate channel images of αp-[WT]-40-mRuby3 and αp-[WT]-80-mRuby3. [Figure 26] Mander coexistence score between converted FDG and the fluorescent reporter. Data analyzed 30 minutes after FDG addition. Background thresholds were automatically set. [Figure 27A-D] The Reinweaver-Burk plots for determining Km and Vmax are shown. Figure 27A, αp-mRuby3; Figure 27B, αp-[WT]-20-mRuby3; Figure 27C, αp-[WT]-40-mRuby3; and Figure 27D, αp-[WT]-80-mRuby3, Reinweaver-Burk plots prepared using various starting concentrations of FDG. The slope (Vo) was determined from fluorescence generation over a 20-minute process. Interference and slope were used in the calculation of Km and Vmax. [Figure 28A-C]The image shows enzyme droplets formed using varying ratios of aromatic to aliphatic residues. All scale bars are 5 μm. Figure 28A shows confocal microscopy images observing the fluorescence conversion of fluorescein di-β-D-galactopyranoside (FDG) in αp-mRuby3, αp-[WT]-40-mRuby3, αp-[3Y7:V7]-40-mRuby3, and αp-[Y7:V7]-40-mRuby3. A decrease in the aromatic:aliphatic ratio did not increase FDG conversion over time, but altered the uptake dynamics using polypeptides with smaller aromatic:aliphatic ratios, observing higher uptake at earlier time points after FDG addition. Figure 28B shows quantitative values ​​of intracellularly converted FDG, standardized against the mRuby3 fluorescence level. Slight differences exist between A-IDPs with different aromatic:aliphatic content ratios. Error bars indicate the standard error of the mean. Figure 28C shows that all αp-A-IDP-mRuby3 fusions exhibit a higher ratio of intracellular FDG fluorescence, indicating longer persistence of fluorescent FDG within the intracellular space compared to the αp-mRuby3 control. Slight differences exist between A-IDPs with different levels of aromatic content. Error bars indicate the standard error of the mean. [Figure 29A-B] The enzymatic activity of αp-[V7]-40-mRuby3 is shown. All scale bars are 5 μm. Figure 29A shows a confocal microscope image showing the fluorescence conversion of fluorescein di-β-D-galactopyranoside (FDG) bound to soluble αp-[V7]-40-mRuby3. Figure 29B shows the intracellular concentration of fluorescein produced by the catalytic conversion of FDG by αp-[V7]-40-mRuby3, standardized against the mRuby3 fluorescence of each individual cell (n≒300). The soluble fusion shows lower levels of enzymatic activity than the αp-A-IDP fusion, which forms all the spots. αp-mRuby3 data in Figure 24, redrawn to show scale. Error bars show the mean standard error. [Figure 30A-D]This paper presents examples of various fusion proteins that are expressed at low levels in prokaryotic expression systems when fused to disordered biomolecules, and demonstrates that recovery into soluble fractions is possible by utilizing the phase separation behavior of biomolecules. This can be done using mAb-binding proteins with nanobody folding structures that bind to mAbs (ZD, Figure 30A); fluorescent fusion proteins with beta-barrel structures (sfGFP, Figure 30B); therapeutic protein peptides with a strong alpha-helical tendency (GLP-1, Figure 30C); RNA-binding proteins with tandem repeat sequence structures (PUMHD, Figure 30D); and antimicrobial peptides that exhibit cytotoxic tendencies in E. coli. [Figure 31] This describes the incubation of mAbs with phase-separated biomacromolecules fused to a domain derived from protein A that binds to mAbs. The biomacromolecule is bound to the mAbs and centrifuged to capture the mAb heavy chain (HC) and light chain (LC). The supernatant from this step is flowed into lanes 2 and 5. The supernatant is then removed, and the pellet is resuspended in a low-pH elution buffer to induce dissociation between the biomacromolecule-ZD fusion and the mAbs. The solution is spun again, thereby obtaining an elution supernatant (lanes 3, 6) containing pure mAb HC and LC as well as other protein contaminants. The elution pellet contains biomacromolecules but not mAbs (lanes 4, 7). [Figure 32] Microscopic images of fluorescently labeled mAbs (red / white on grayscale) visualized in the presence of a phase-separated protein ((GRGDQPYQ)40, SEQ ID NO: 3 with m=40) fused to the Z-domain of protein A(ZD) are shown. The coexistence of the droplet's fluorescence signal is observable in the first image. When the buffer pH is lowered at t=0, a reversal of the fluorescence signal occurs, suggesting that the mAb completely dissociated from the phase-separated protein-ZD fusion protein (white arrow) and entered the surrounding solution (red). Since the droplet fusion occurs in 60–240 seconds (arrow), these biomolecules-fusion proteins retain their liquid-like behavior. [Figure 33]This shows the expression of fusion proteins containing various AMPs, both unfused (left) and fused to various biomolecules (right). When the fusion protein is not expressed (top), cell growth proceeds as standard, as measured by gradual absorption at OD600. When AMP alone is expressed (bottom-left), cell growth is inhibited (the growth curve is shifted to a later time). When the AMP-biomolecule fusion protein is expressed, standard growth is restored, suggesting a reduction in AMP availability. [Figure 34] This describes an injection strategy for forming a subcutaneous depot in vivo. Injection using a solubilizer such as urea allows injection under ambient conditions. Because the solubilizer diffuses faster than the polypeptide, the solvent becomes a poor solvent, and the polypeptide separates into phases. Alternatively, the dehydrated coacervate is implanted into the subcutaneous space and indirectly slowly rehydrates into a two-phase regime. [Figure 35] This image shows fluorescence molecular tomography of (GRGDSPYQ)40 labeled with a near-infrared fluorescent dye after injection in the presence of 2M urea + PBS. The injection concentration is 175 μm, corresponding to 1.2 mg of total protein. [Figure 36] The image shows fluorescence molecular tomography of (GRGDSPYQ)40 labeled with a near-infrared fluorescent dye after injection in a dehydrated state. The injection mass is equivalent to 1.2 mg of total protein. [Figure 37] This shows the binodal phase boundary of GLP-1-RIDP with a smaller molecular weight. Data were collected in 140 mM PBS. The dotted line is an approximation line for y = m*ln(x) + b. [Figure 38] This image shows the blood glucose levels of a GLP-1-RIDP fusion protein with a size of approximately 20 kDa and variable Csat. Data were collected from C57Bl / 6J mice fed a 60% fat diet. Error bars represent the standard error of the mean (n=5). [Figure 39]Body weight changes in mice carrying a subcutaneous GLP-1-RIDP depot with a size of approximately 20 kDa and variable Csat. Data collected from C57Bl / 6J mice fed a 60% fat diet. Error bars represent the standard deviation of the mean (n=5). [Figure 40] This shows the binodal phase boundary of GLP-1-RIDP with a larger molecular weight. Data were collected in 140 mM PBS. The dotted line is an approximation line for y = m*ln(x) + b. [Figure 41] This image shows the blood glucose levels of a GLP-1-RIDP fusion protein with a size of approximately 35 kDa and variable Csat. Data were collected from C57Bl / 6J mice fed a 60% fat diet. Error bars represent the standard error of the mean (n=5). [Figure 42] Although they have different molecular weights, these GLP-1-RIDP fusion proteins of Csat show blood glucose levels. Data were collected from C57Bl / 6J mice fed a 60% fat diet. Error bars represent the standard error of the mean (n=5). [Figure 43] This shows the body weight changes of mice carrying a subcutaneous GLP-1-RIDP depot with a size of approximately 35 kDa and variable Csat. Data were collected from C57Bl / 6J mice fed a 60% fat diet. Error bars represent the standard error of the mean (n=5). [Modes for carrying out the invention]

[0007] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any conflict, this document shall prevail, including the definitions. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are for illustrative purposes only and are not intended to limit the scope of this invention.

[0008] As used herein, the terms “comprise” (including the third-person singular), “include” (including the third-person singular), “have,” “has,” “can,” “contain” (including the third-person singular), and their variations are intended to be unrestricted transitional phrases, terms, or statements that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include multiple references unless the context clearly indicates otherwise. This disclosure also intends that other embodiments, whether expressly stated or not, “include,” “consist of,” and “essentially consist of” embodiments or components presented herein. For the purpose of detailing numerical ranges in this specification, numbers that fall between each other are explicitly intended with the same degree of precision. For example, for the range 6–9, the numbers 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0009] When applied to one or more values ​​and used herein, the term “about” refers to values ​​similar to the reference value mentioned. In certain embodiments, unless otherwise specified or evident from the context, the term “about” refers to a range of values ​​that are not 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction of the given reference value (except where such numbers exceed 100% of the possible values). "Affinity" refers to the strength of the binding of a binding polypeptide to its target (i.e., its binding partner).

[0010] An "agonist" refers to an entity that binds to a receptor, activates the receptor, and produces a biological response. An "antagonist" blocks or inhibits the action or signaling of an agonist. An "inverse agonist" produces the opposite effect of an agonist. The activity of agonists, antagonists, and inverse agonists can be determined in vitro, in sights, in vivo, or a combination thereof. As used herein, “amino acids” refers to naturally occurring amino acids and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids may be referred herein by either their commonly known three-letter or one-letter notation, as recommended by the IUPAC-IUB biochemical nomenclature. Amino acids include side chains and polypeptide backbone portions.

[0011] As used herein, the term “biomarker” refers to a naturally occurring biological molecule present in a subject at various concentrations useful for identifying and / or classifying a disease or condition. Biomarkers may include genes, proteins, polynucleotides, nucleic acids, ribonucleic acids, polypeptides, or other biological molecules used as indicators or markers of disease. In some embodiments, the biomarker includes a disease marker. For example, the biomarker may be a gene that is upregulated or downregulated in a subject having a disease. As another example, the biomarker may be a polypeptide whose levels increase or decrease in a subject having a disease or being at risk of developing a disease. In some embodiments, the biomarker includes a small molecule. In some embodiments, the biomarker includes a polypeptide.

[0012] The terms “control,” “reference level,” and “reference” are used interchangeably herein. A reference level may be a predetermined value or range and is used as a criterion for evaluating measured results. Where used herein, “control group” refers to the control group. A predetermined level may be a cutoff value derived from the control group. A predetermined level may be a mean derived from the control group. The cutoff value (or predetermined cutoff value) may be determined by an adaptive index model (AIM) methodology. The cutoff value (or predetermined cutoff value) may be determined by a patient operating curve (ROC) analysis derived from biological samples of the patient group. ROC analysis, commonly known in the biological field, is the determination of the ability of a test to distinguish one condition from another, for example, to determine the performance of each marker in identifying patients with CRC. A description of ROC analysis is provided in PJ Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is incorporated herein by reference in its entirety. Alternatively, the cutoff value may be determined by quartile analysis of biological samples from the patient group. For example, the cutoff value may be determined by selecting a value corresponding to any value in the 25th–75th percentile range, preferably a value corresponding to the 25th, 50th, or 75th percentile, more preferably a value corresponding to the 75th percentile. Such statistical analysis may be performed using any method known in the art and can be carried out through any number of commercially available software packages (e.g., Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC). Healthy or normal levels or ranges for target or protein activity may be defined according to standard practices. The term “expression vector” refers to a plasmid, virus, or other medium known in the art into which a nucleic acid sequence encoding a desired protein is inserted or introduced. The term "host cell" refers to a cell that is readily transformed, transfected, transduced, or bound to a nucleic acid construct or expression vector. Host cells can be derived from plants, bacteria, yeasts, fungi, insects, animals, and the like. In some embodiments, the host cell includes Escherichia coli.

[0013] As used herein, “polymer” is intended to include homopolymers, heteropolymers, block polymers, copolymers, ter-polymers, and the like, as well as blends, combinations, and mixtures thereof. Examples of polymers include, but are not limited to, functionalized polymers, such as polymers containing 5-vinyltetrazole monomer units and having a molecular weight distribution of less than 2.0. A polymer may be one or more crosslinked structures, such as star-block copolymers, linear polymers, branched polymers, highly branched polymers, dendritic polymers, comb polymers, graft polymers, brush polymers, bottlebrush copolymers, and block copolymers containing blocks of 5-vinyltetrazole monomer units. A polymer includes, but is not limited to, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, polystyrenes, polynorbornene, and monomers having unsaturated bonds. For example, amphiphilic comb polymers are described in Mayes et al., U.S. Patent Application Publication No. 2007 / 0087114 and U.S. Patent No. 6,207,749, the respective disclosures of which are incorporated herein by reference in whole. The amphiphilic comb-type polymer may exist in the form of a copolymer containing a backbone formed from a hydrophobic, water-insoluble polymer and side chains formed from short, hydrophilic, non-cell-binding polymers.Other examples of polymers include polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; polyvinyl halides such as polyvinyl acetate and polyvinyl chloride; polysiloxanes; polystyrene; polyurethane; polyacrylates; for example, poly(methyl(meth)acrylate), poly(ethyl(meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(tert-butyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl(methyl) The polymers include, but are not limited to, polyacrylamides such as poly(acrylate), poly(isopropylacrylate), poly(isobutylacrylate), and poly(octadecylacrylate); poly(acrylamide), poly(methacrylamide), poly(ethylacrylamide), poly(ethylmethacrylamide), poly(N-isopropylacrylamide), and poly(n, iso, and tert-butylacrylamide); and copolymers and mixtures thereof. These polymers may include useful derivatives, including substitutions, addition of chemical groups such as alkyl groups and alkylene groups, hydroxylation, oxidation, and other modifications routinely performed by those skilled in the art. The polymers may include zwitterionic polymers such as polyphospholycoline, polycarboxybetaine, and polysulfobetaine. The polymers may have side chains of betaine, carboxybetaine, sulfobetaine, oligoethylene glycol (OEG), sarcosine, or polyethylene glycol (PEG). For example, poly(oligoethylene glycol methacrylate) (poly(OEGMA)) may be used. Poly(OEGMA) may be hydrophilic, water-soluble, non-fouling, non-toxic, and non-immunogenic due to the OEG side chains.

[0014] As used herein, “polynucleotide” may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Polynucleotides may be nucleic acids, natural or synthetic DNA, genomic DNA, cDNA, RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides may be obtained by chemical synthesis or by recombinant methods.

[0015] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. Polypeptides can be native, synthetic, modified, or a combination of native and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to the locally ordered three-dimensional structure within a polypeptide. These structures are commonly known as domains, e.g., enzyme domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are parts of a polypeptide that form smaller units of the polypeptide and are typically 15 to 350 amino acid long. Typical domains include those with enzymatic activity or ligand-binding activity. Typical domains consist of sections of less organized structures, such as beta-sheets and alpha-helical extensions. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three-dimensional structure formed by non-covalent bonds of independent tertiary units.

[0016] The terms “reporter,” “reporter group,” “label,” and “detectable label” are used interchangeably herein. A reporter has the ability to produce a detectable signal. A label can produce a signal that is detectable by visual or instrumental means. Various reporter groups can be used, differing in the physical properties of the signal transduction (e.g., fluorescence, electrochemistry, nuclear magnetic resonance (NMR), and electromagnetic response (EPR)) and the chemical properties of the reporter group. Various reporters include signal-generating substances such as chromatens, fluorescent compounds, chemiluminescent compounds, and radioactive compounds. In some embodiments, the reporter includes a radiolabel. The reporter may include a light-producing moiety, e.g., an acridinium compound, and a fluorescence-producing moiety, e.g., fluorescein. In some embodiments, the signal from the reporter is a fluorescent signal. The reporter may include a fluorophore. Examples of fluorophores include, but are not limited to, acrylodane (6-acryloy-1-2-dimethylaminonaphthalene), badan (6-bromo-acetyl-2-dimethylaminonaphthalene), rhodamine, naphthalene, danzyladiridine, 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenzo-2-oxa-1,3-diazole (IANBDE), 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenzo-2-oxa-1,3-diazole (IANBDA), fluorescein, dipyromethaneborone difluoride (BODIPY), 4-nitrobenzo[c][1,2,5]oxadiazole (NBD), Alexa fluorescent dyes, and their derivatives. Fluorescein derivatives may include, for example, 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein, and isothiocyanates.

[0017] As used herein, "sample" or "test sample" can mean any sample in which the presence and / or level of a target is to be detected or determined. The sample can include a liquid, solution, emulsion, or suspension. The sample can include a medical sample. The sample can include any biological fluid or tissue such as blood, whole blood, fractions of blood such as plasma or serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage fluid, vomit, excrement, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or a combination thereof. In some embodiments, the sample includes an aliquot. In other embodiments, the sample includes a biological fluid. The sample can be obtained by any means known in the art. The sample can be obtained from a patient and used directly or can be pretreated by filtration, distillation, extraction, concentration, centrifugation, interaction with interfering components, addition of reagents, etc., to be considered herein or otherwise modify the characteristics of the sample in some ways known in the art.

[0018] As used herein, the term "sensitivity" refers to the number of true positives divided by the number of true positives plus the number of false negatives, and the sensitivity ("sens") can be in the range of 0 < sens < 1. Ideally, embodiments of the methods herein have a number of false negatives equal to or approximately zero, and thus there are no subjects who are incorrectly identified as not having the disease when they actually have the disease. Conversely, the assessment, which is a complementary measure of sensitivity, is generally made from the ability of a predictive algorithm to correctly classify negatives.

[0019] As used herein, the term "specificity" refers to the number of true negatives divided by the number of true negatives plus the number of false positives, and the specificity ("spec") can be in the range of 0 < spec < 1. Ideally, the methods herein have a number of false positives equal to or nearly zero, and thus there are no subjects misidentified as having a disease when they actually do not have the disease. Thus, a method having both a sensitivity and a specificity equal to 1, or 100%, is preferred. By "specifically binds," it is generally meant that a polypeptide binds to a target more rapidly than it binds to a random unrelated target.

[0020] As used herein, "subject" can mean a mammal that desires or requires a peptide biopolymer described herein, including one or more fusion proteins. The subject can be a human or non-human animal. The subject can be a mammal. The mammal can be a primate or non-primate. The mammal can be a primate such as a human; non-primates such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primates such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject can be of any age or stage of development, such as, for example, an adult, juvenile, or infant.

[0021] "Transition" or "phase transition" refers to the aggregation of a thermoresponsive polypeptide. The phase transition is the lower critical solution temperature (LCST) or the inverse transition temperature T ^This is called an inverse transition cycle, which occurs rapidly and reversibly at a specific temperature. Below the transition temperature, the thermoresponsive polypeptide (or polypeptides containing a thermoresponsive polypeptide) is highly soluble. Upon heating above the transition temperature, the thermoresponsive polypeptide hydrophobically disintegrates and aggregates, thereby forming separate gel-like phases. "Inverse transition cycle" refers to a protein purification method for thermoresponsive polypeptides (or polypeptides containing a thermoresponsive polypeptide). The protein purification method may include cycling the solution through a soluble phase and an insoluble phase, thereby utilizing the reversible phase transition behavior of the thermoresponsive polypeptide to remove contaminants. When referring to protection from a disease, “treatment” or “treatment” means preventing, suppressing, inhibiting, restoring, or eliminating the disease. Preventing a disease includes administering the composition of the present invention to a subject before the onset of the disease. Suppressing a disease includes administering the composition of the present invention to a subject after the induction of the disease but before the appearance of its clinical manifestations. Inhibiting or restoring a disease includes administering the composition of the present invention to a subject after the appearance of its clinical manifestations.

[0022] "Substantially identical" may mean that the first and second amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% across a range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acid regions. As used herein, “valence” refers to a possible bonding unit or bonding site. The term “polyvalent” refers to multiple possible bonding units. The terms “multimer” and “polyvalent” are used interchangeably herein. With respect to polynucleotides, as used herein, “variant” means (i) a portion or fragment of a reference nucleotide sequence; (ii) a reference nucleotide sequence or a complementary chain to a portion thereof; (iii) a polynucleotide substantially identical to a reference polynucleotide or its complementary chain; or (iv) a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide, its complementary chain, or a sequence substantially identical thereto.

[0023] A “variant” can be further defined as a peptide or polypeptide that has a different amino acid sequence due to an amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. Typical examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide, or the ability to promote an immune response. A variant may mean a substantially identical sequence. A variant may mean its functional fragment. A variant may also mean multiple copies of a polypeptide. Multiple copies may be tandem or separated by a linker. A variant may also mean a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substituting an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree of charged region, and distribution), are typically recognized in the art as involving only minor changes. These minor changes can be identified in part by considering the hydrophobicity and hydrophilicity indicators of amino acids. See Kyte et al., J. Mol. Biol. 1982, 757, 105-132. The hydrophobicity and hydrophilicity index of amino acids is based on consideration of their hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity and hydrophilicity indexes can be substituted to retain protein function. In one embodiment, amino acids with hydrophobicity and hydrophilicity indexes of ±2 are substituted. The hydrophobicity of amino acids can also be used to identify substitutions that result in polypeptides that retain biological function. Consideration of the precursors of the polypeptide's hydrophilicity allows for the calculation of the polypeptide's maximum local mean hydrophilicity, a useful measurement that has been reported to be well related to antigenicity and immunogenicity, as discussed in U.S. Patent No. 4,554,101, incorporated herein by reference. Substitution of amino acids with similar hydrophilicity values ​​can result in polypeptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other.Both the hydrophobic and hydrophilic values ​​of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions equivalent to biological function are based on the relative similarity of amino acids, particularly their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0024] A variant may be a polynucleotide sequence that is substantially identical across the entire gene sequence or a fragment thereof. A polynucleotide sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical across the entire gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical across the entire amino acid sequence or a fragment thereof. An amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical across the entire amino acid sequence or a fragment thereof.

[0025] Fusion protein As used herein, the term “fusion protein” means at least one essentially disordered polypeptide and at least one other polypeptide. The fusion protein may optionally include at least one linker. In one embodiment, the essentially disordered polypeptide has controlled reversible phase separation.

[0026] In some embodiments, the fusion protein comprises one or more polypeptides having controlled reversible phase separation. A polypeptide having controlled reversible phase separation may comprise multiple repeat sequences of a peptide motif. The fusion protein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 40, at least 60, at least 80, at least 120, at least 160, or at least 200 polypeptides having controlled reversible phase separation or repeat sequences of peptide motifs having controlled reversible phase separation. The fusion protein may comprise less than 30, less than 25, or less than 20 repeat sequences of polypeptides having controlled reversible phase separation or peptide motifs having controlled reversible phase separation. The fusion protein may contain 1 to 160, 1 to 80, 1 to 60, 1 to 40, 1 to 20, or 1 to 10 repeat sequences of polypeptides or peptide motifs having controlled reversible phase separation. In such embodiments, the polypeptides having controlled reversible phase separation may be the same as or different from each other. In some embodiments, the fusion protein contains one or more polypeptides (e.g., repeat sequences of peptide motifs) having controlled reversible phase separation located in tandem with each other.

[0027] In some embodiments, the fusion protein comprises one or more binding polypeptides. The fusion protein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 binding polypeptides. The fusion protein may comprise less than 30, less than 25, less than 20, less than 10, or less than 5 binding polypeptides. The fusion protein may comprise 1 to 30, 1 to 20, or 1 to 10 binding polypeptides. In such embodiments, the binding polypeptides may be the same or different from each other. In some embodiments, the fusion protein comprises one or more binding polypeptides located in tandem with each other. In some embodiments, the fusion protein comprises 2 to 6 binding polypeptides. In some embodiments, the fusion protein comprises 2 binding polypeptides. In some embodiments, the fusion protein comprises 3 binding polypeptides. In some embodiments, the fusion protein comprises 4 binding polypeptides. In some embodiments, the fusion protein contains five binding polypeptides. In some embodiments, the fusion protein contains six binding polypeptides.

[0028] The fusion protein may be recombinantly expressed in host cells as described by those skilled in the art. The fusion protein may be purified by any means known to those skilled in the art. For example, the fusion protein may be purified using chromatography such as liquid chromatography, size exclusion chromatography, or affinity chromatography, or a combination thereof. In some embodiments, the fusion protein is purified without chromatography. In some embodiments, the fusion protein is purified using an inverse transfer cycle.

[0029] polypeptides with controlled, reversible phase separation Polypeptides having controlled reversible phase separation may include any polypeptide having or not having minimal secondary structure, as observed by CD, including a repeating amino acid sequence, and being soluble at temperatures below its lower critical solubility temperature (LCST) and / or above its upper critical solubility temperature (UCST). LCST is the temperature below which the polypeptide is miscible. UCST is the temperature above which the polypeptide is miscible. In some embodiments, polypeptides having controlled reversible phase separation exhibit only UCST behavior. In some embodiments, polypeptides having controlled reversible phase separation exhibit only LCST behavior. In some embodiments, polypeptides having controlled reversible phase separation exhibit both UCST and LCST behavior. Polypeptides having controlled reversible phase separation may include repeating amino acid sequences. Polypeptides having controlled reversible phase separation may have LCSTs of about 0°C to about 100°C, about 10°C to about 50°C, or about 20°C to about 42°C. Polypeptides having controlled reversible phase separation may have a UCST of about 0°C to about 100°C, about 10°C to about 50°C, or about 20°C to about 42°C. In some embodiments, polypeptides having controlled reversible phase separation have a transition temperature of room temperature (about 25°C) to body temperature (about 37°C). In some embodiments, a fusion protein comprising one or more thermoresponsive polypeptides has a transition temperature of room temperature (about 25°C) to body temperature (about 37°C). In some embodiments, polypeptides having controlled reversible phase separation do not exhibit LCST or UCST behavior. Polypeptides having controlled reversible phase separation may have an LCST or UCST below or above body temperature at concentrations administered to a subject, as peptide biomacromolecules comprising one or more fusion proteins. In some embodiments, the polypeptide having controlled reversible phase separation comprises one or more thermoresponsive polypeptides. The thermoresponsive polypeptides may include, for example, elastin-like polypeptides (ELPs) and resilin-like proteins (RLPs).

[0030] In some embodiments, a polypeptide having controlled reversible phase separation comprises a plurality of polypeptides having controlled reversible phase separation. In one embodiment, the polypeptide having controlled reversible phase separation is a diblock of two or more polypeptides having controlled reversible phase separation. In one embodiment, the polypeptide having controlled reversible phase separation comprises a diblock of resilin-like proteins (RLPs) and elastin-like polypeptides (ELPs).

[0031] In one embodiment, a polypeptide having controlled reversible phase separation comprises one or more core polypeptides. In one embodiment, the core polypeptide is a resilin-like polypeptide (RLP). The RLP is derived from arthropod Rec1-resilin. Rec1-resilin is environmentally responsive and exhibits two-phase transition behavior. Thermoresponsive RLPs may have LCST and UCST. Further examples of suitable thermoresponsive polypeptides are described in U.S. Patent Application Publications 2012 / 0121709 and 2015 / 0112022, which are incorporated herein by reference, respectively. In one embodiment, the RLP polypeptide is sequence (GRGDSPYS) n (Sequence ID 1) is included. Polypeptides with controlled reversible phase separation are (G1-R2-G3-D4-S5-P6-Y7-S8). n The formula may include an amino acid sequence containing (wherein n is 20 to 200). In some embodiments, n is 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300. In some embodiments, n may be less than 500, less than 400, less than 300, less than 200, or less than 100. In some embodiments, n may be 1 to 500, 1 to 400, 1 to 300, or 1 to 200. In some embodiments, n is 20, 40, 60, 80, 100, 120, 160, 180, or 200. In one embodiment, n is a repeating sequence of 20 to 200. In another embodiment, n is a repeating sequence of 20 to 60.

[0032] The thermoresponsive polypeptide may have a phase transition. The thermoresponsive polypeptide may impart phase transition characteristics to an unstructured polypeptide or fusion protein. "Phase transition" or "transition" may refer to the aggregation of a thermoresponsive polypeptide that occurs rapidly and reversibly at a specific temperature, called the lower critical solution temperature (LCST) or inverse transition temperature (T t ). Below the transition temperature (LCST or T t ), the thermoresponsive polypeptide (or polypeptide containing the thermoresponsive polypeptide) may be highly soluble. Upon heating above the transition temperature, the thermoresponsive polypeptide becomes hydrophobic, collapses, and aggregates, thereby forming distinct gel-like phases.

[0033] The thermoresponsive polypeptide may undergo phase transitions at various temperatures and concentrations. The thermoresponsive polypeptide may, for example, not affect the binding or effectiveness of a binding polypeptide. The thermoresponsive polypeptide may enable a fusion protein to be cleaved by the user into any number of desired transition temperatures, molecular weights, and formats.

[0034] The thermoresponsive polypeptide exhibits inverse phase transition behavior, and thus, a fusion protein containing the thermoresponsive polypeptide may exhibit inverse phase transition behavior. Inverse phase transition behavior may be used to form a drug depot within a target tissue for slow (delayed) release of the fusion protein. Inverse phase transition behavior may also enable purification of the fusion protein using an inverse transition cycle, thereby potentially eliminating the need for chromatography.

[0035] One embodiment described herein is (X-Z1-X-Z2-Z3-X-Z4-Z3) n (wherein X is proline (P) or glycine (G), and the ratio of P:G is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D is any number, and the ratio of K:R may be any number; Z2 is composed of Asp(D), Arg(R), and Glu(E), where the ratio R:D can be any number, and the ratio D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number; Z4 is a polypeptide having controlled reversible phase separation, comprising 10 or more repeat sequences of amino acid sequences (where Y:H:W:F:M:V:I:A:L can be any number), including tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L).

[0036] In one embodiment, X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. In another embodiment, Z1 is arginine (R), aspartic acid (D), or lysine (K), and the R:D ratio is not greater than 1:5, and the K:R ratio can be any number. In another embodiment, phase separation depends on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration. In another embodiment, n is 10 to 200. In another embodiment, the molecular weight is at least 5 kDa to 500 kDa. In another embodiment, the molecular weight is about 5 kDa to about 100 kDa. In another embodiment, the phase separation temperature is 0 to 100°C. In another embodiment, the phase separation temperature is 4 to 25°C, about 25°C, 25 to 37°C, about 37°C, 35 to 38°C, or >38°C. In another embodiment, the polypeptide comprises a modified amino acid, a reporter protein, or an enzyme. In another embodiment, the sequence is (GRGDSPYS) m(wherein m is 20 to 80) includes. In another embodiment, polypeptide includes sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, Includes an array selected from 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279, or one or more combinations thereof.

[0037] [Table 1] JPEG2026091842000002.jpg219154 JPEG2026091842000003.jpg219154 TIFF2026091842000004.tif219154 JPEG2026091842000005.jpg219154 JPEG2026091842000006.jpg219154 JPEG2026091842000007.jpg224154 JPEG2026091842000008.jpg230154 JPEG2026091842000009.jpg219154 JPEG2026091842000010.jpg219154 JPEG2026091842000011.jpg208154 JPEG2026091842000012.jpg203154 JPEG2026091842000013.jpg219154 JPEG2026091842000014.jpg208154 JPEG2026091842000015.jpg229154 JPEG2026091842000016.jpg219154 JPEG2026091842000017.jpg208154 JPEG2026091842000018.jpg208154 JPEG2026091842000019.jpg208154 JPEG2026091842000020.jpg192154 JPEG2026091842000021.jpg208154 JPEG2026091842000022.jpg224154 JPEG2026091842000023.jpg214154 JPEG2026091842000024.jpg219154 JPEG2026091842000025.jpg219154 JPEG2026091842000026.jpg219154 JPEG2026091842000027.jpg229154 JPEG2026091842000028.jpg181153

[0038] Combined polypeptides The conjugating polypeptide (or “targeting polypeptide”) may contain any polypeptide capable of binding to at least one target. The conjugating polypeptide may bind to at least one target. The “target” may be an entity to which the conjugating polypeptide is capable of binding. The target may include, for example, another polypeptide, a cell surface receptor, a carbohydrate, an antibody, a small molecule, or a combination thereof. The target may be a biomarker. The target may be activated via agonism or blocked via antagonistism. The conjugating polypeptide may bind specifically to the target. By binding to the target, the conjugating polypeptide may act as a targeting moiety, an agonist, an antagonist, or a combination thereof. In some embodiments, the conjugating polypeptide domain binds.

[0039] The binding polypeptide may be a monomer that binds to a target. The monomer may bind to one or more targets. The binding polypeptide may form an oligomer. The binding polypeptide may form an oligomer having the same or different binding polypeptides. The oligomer may bind to a target. The oligomer may bind to one or more targets. One or more monomers in the oligomer may bind to one or more targets. In some embodiments, the fusion protein is polyvalent. In some embodiments, the fusion protein binds to multiple targets. In some embodiments, the activity of the binding polypeptide alone is the same as the activity of the binding protein when it is a part of the fusion protein.

[0040] In one embodiment, the binding polypeptide is an antimicrobial peptide selected from the following: staphylococcus protein A(ZD)-derived antibody-binding domain (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), cathelicidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 3 9); Protein drugs, GLP-1 (SEQ ID NO: 177); Fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or one or more AAV-binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195).

[0041] Linker In some embodiments, the fusion protein further comprises at least one linker. In some embodiments, the fusion protein comprises one or more linkers. In such embodiments, the linkers may be the same as or different from one another. The fusion protein may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 linkers that do not contain linkers. The fusion protein may contain less than 500, less than 400, less than 300, or less than 200 linkers. The fusion protein may contain 1-1000, 10-900, 10-800, or 5-500 linkers.

[0042] The linker may be located between a bound polypeptide and a polypeptide having controlled reversible phase separation, between bound polypeptides, between polypeptides having controlled reversible phase separation, or in combination thereof. Multiple linkers may be located adjacent to one another. Multiple linkers may be located adjacent to one another between a bound polypeptide and a polypeptide having controlled reversible phase separation.

[0043] The linker may be a polypeptide of any amino acid sequence and length. The linker may act as a spacer peptide. The linker may arise between polypeptide domains. The linker can sufficiently separate the binding domain of the binding polypeptide while preserving the activity of the binding domain. In some embodiments, the linker contains a charged amino acid. In some embodiments, the linker is mobile. In some embodiments, the linker contains at least one glycine and at least one serine. In some embodiments, the linker contains at least one proline.

[0044] Polynucleotides Polynucleotides encoding the fusion proteins detailed herein are further provided. The vector may also contain the polynucleotides encoding the fusion proteins detailed herein. To obtain polypeptide expression, typically the polynucleotide encoding the polypeptide is subcloned into an expression vector containing a promoter for transcription, a transcription / translation terminator, and, in the case of nucleic acids encoding proteins, a promoter for directing the ribosome binding site for transcription initiation. An example of a vector is pET24. Suitable bacterial promoters are well known in the art. Host cells transformed or transfected with an expression vector containing the polynucleotide encoding the fusion proteins detailed herein are further provided. Bacterial expression systems for expressing proteins are available, for example, in Escherichia coli, Bacillus species, and Salmonella (Paiva et al., Gene 1983, 22, 229-235; Mosbach et al., Nature 1983, 302, 543-545). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known and commercially available in the art. Retroviral expression systems can be used in the present invention. In some embodiments, the fusion protein is SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, Includes one or more repeating sequences or single sequences of 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279.In some embodiments, the fusion protein is sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 , comprising one or more repeat sequences or single sequences of a polypeptide encoded by any one polynucleotide sequence of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158. In some embodiments, the fusion protein comprises a polypeptide comprising any one amino acid sequence of SEQ ID NOs: 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, or 316.

[0045] Administration Peptide biopolymers comprising one or more fusion proteins, as detailed herein, can be formulated according to standard techniques well known to those skilled in the pharmaceutical field to form therapeutic agents or targeted delivery agents. Such compositions comprising peptide biopolymers comprising one or more fusion proteins can be administered according to techniques well known to those skilled in the pharmaceutical field, taking into account factors such as dosage and age, sex, weight, the condition of the specific subject, and route of administration. Peptide biopolymers containing one or more fusion proteins can be administered prophylactically or therapeutically. In prophylactic administration, the peptide biopolymer may be administered in an amount sufficient to induce a response. In therapeutic application, the peptide biopolymer is administered to the target in need in an amount sufficient to induce a therapeutic effect. The amount sufficient to achieve this is defined as the "therapeutic effective dose." The effective dose for this use depends, for example, on the specific composition of the peptide biopolymer regime being administered, the mode of administration, the stage and severity of the disease, the patient's general health condition, and the physician's judgment.

[0046] Peptide biopolymers can be administered by methods well known in the art, as described in Donnelly et al. Ann. Rev. Immunol. 1997, 75, 617-648; Feigner et al., U.S. Patent No. 5,580,859; Feigner, U.S. Patent No. 5,703,055; and Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference in their entirety. Peptide biopolymers can be complexed into particles or beads that can be administered to an individual, for example, using a vaccine gun. Those skilled in the art will know that the selection of a pharmaceutically acceptable carrier containing physiologically acceptable compounds depends, for example, on the route of administration. Peptide biopolymers can be delivered via various routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intra-arterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, peptide biopolymers are administered intravenously, intra-arterial, or intraperitoneally to the target.

[0047] Peptide biopolymers may be liquid preparations such as suspensions, syrups, or elixirs. Peptide biopolymers can be incorporated into liposomes, microparticles, or other polymer matrices (for example, by the method described herein, which is incorporated by reference in whole, Feigner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993)). Liposomes may consist of phospholipids or other lipids and may be non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0048] In some embodiments, the peptide biopolymer is administered in a controlled-release formulation. In some embodiments, the peptide biopolymer comprises one or more thermoresponsive polypeptides having a transition temperature, such that the peptide biopolymer remains soluble before administration and transfers upon administration to a gel-like depot in the subject. In some embodiments, the peptide biopolymer comprises one or more fusion proteins comprising one or more thermoresponsive polypeptides having a transition temperature, such that the fusion protein remains soluble at room temperature and transfers upon administration to a gel-like depot in the subject. For example, in some embodiments, the fusion protein comprises one or more thermoresponsive polypeptides having a transition temperature between room temperature (about 25°C) and body temperature (about 37°C), thereby allowing the fusion protein to be administered to form a depot. As used herein, “depot” refers to a gel-like composition containing a fusion protein that releases the fusion protein over time. In some embodiments, the peptide biopolymer can be injected subcutaneously or intratumorally to form a depot (coacervate). Depots can result in the delayed release of peptide biomacromolecules. Depots can result in the slow release of peptide biomacromolecules into the circulation or tumors, etc. In some embodiments, peptide biomacromolecules may be released from the depot over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3.5 weeks, at least about 4 weeks, or at least about 1 month.

[0049] detection As used herein, the terms “detect” or “determine presence” refer to the qualitative measurement of one or more peptide biopolymers, targets, or peptide biopolymers bound to targets at undetectable low, standard, or high concentrations. Detection may include in vitro, ex vivo, or in vivo detection. Detection may include detecting the presence of one or more peptide biopolymers containing one or more peptide biopolymers or targets versus the absence of one or more peptide biopolymers or targets. Detection may also include the quantification of levels of one or more peptide biopolymers or targets. The terms “quantify” or “quantify” may be used interchangeably and refer to the process of determining the amount or abundance of a substance (e.g., peptide biopolymer or target), whether relative or absolute. Any suitable detection method is within the general scope of this disclosure. In some embodiments, the peptide biopolymer includes a reporter bound to it for detection. In some embodiments, the peptide biopolymer is labeled using the reporter. In some embodiments, the detection of target-bound peptide biomolecules may be determined by methods including, but not limited to, band intensity in Western blotting, flow cytometry, radiolabeling imaging, cell binding assays, activity assays, SPR, and immunoassays, or by various other methods known in the art.

[0050] In some embodiments, including those in which the peptide biomolecule is an antibody mimetic for binding to and / or detecting a target, any immunoassay may be used. The immunoassay may be an enzyme-conjugated immunoassay (ELISA), a radioimmunoassay (RIA), a competitive inhibition assay such as a forward or reverse competitive inhibition assay, a fluorescence-spectrochemical assay, or a competitive binding assay. The ELISA may be a sandwich ELISA. Specific immunological binding of the peptide biomolecule to a target can be detected via direct labeling bound to the peptide biomolecule or via indirect labeling such as alkaline phosphatase or horseradish peroxidase. The use of immobilized peptide biomolecules may be incorporated into the immunoassay. The peptide biomolecules may be immobilized on various supports such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), or pieces of solid substrate material. Assay strips can be prepared by coating an array of peptide biomolecules or multiple peptide biomolecules on a solid support. The strip is then immersed in a test biological sample and rapidly processed through a washing and detection process to produce a measurable signal, such as a colored spot.

[0051] Treatment methods for diseases The present invention relates to a method for treating a disease in a subject that requires it. The method may include administering to the subject an effective amount of a peptide biopolymer comprising one or more peptide biopolymers described herein. The disease may be selected from cancer, metabolic diseases, autoimmune diseases, cardiovascular diseases, and orthopedic disorders. In some embodiments, the disease is a disease associated with at least one target of the bound polypeptide. Metabolic disorders can occur when abnormal chemical reactions in the body alter normal metabolic processes. Metabolic disorders may include, for example, insulin resistance, non-alcoholic fatty liver disease, type 2 diabetes, insulin resistance disorders, cardiovascular diseases, arteriosclerosis, lipid-related metabolic disorders, hyperglycemia, hyperinsulinemia, hyperlipidemia, and glucose metabolic disorders.

[0052] Autoimmune diseases result from an abnormal immune response of the body to substances and tissues normally present in the body. Autoimmune diseases include, but are not limited to, lupus, rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, myasthenia gravis, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, Goodpasture syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, polyarteritis nodosa, myocarditis, psoriasis, celiac disease, Crohn's disease, ulcerative colitis, and fibromyalgia. Cardiovascular disease is a type of disease affecting the heart or blood vessels. Cardiovascular disease may include, for example, coronary artery disease (CAD), such as angina pectoris and myocardial infarction (heart attack), stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, and venous thrombosis.

[0053] Orthopedic or musculoskeletal disorders are injuries or pain in the joints, ligaments, muscles, nerves, tendons, and structures supporting the limbs, neck, and back of the body. Orthopedic disorders may include degenerative diseases and inflammatory conditions that cause pain and impair normal activity. Orthopedic disorders may include, for example, carpal tunnel syndrome, epicondylitis, and tendinitis. Cancer may include, but is not limited to, breast cancer, colorectal cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, stomach cancer, esophageal cancer, sarcoma, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphatic system cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, kidney cancer, bladder cancer, thyroid cancer, bone cancer, carcinoma, sarcoma, and soft tissue cancer. In some embodiments, cancer is colorectal cancer. In some embodiments, cancer is colorectal adenocarcinoma.

[0054] One application of protein therapy is cancer treatment. In certain embodiments, the present invention provides a method for using scaffold proteins in the development of antibody mimes for oncological targets of interest. With regard to the emergence of scaffold proteins, manipulation has the potential to design potent protein drugs that are not hindered by steric and structural limitations. While potent protein drugs can be extremely valuable for diagnosis or treatment, effective delivery to the target region can present significant challenges.

[0055] Methods of diagnosing diseases A method for diagnosing a disease is provided herein. The method may include administering a peptide biomolecule containing one or more fusion proteins described herein to a subject and detecting the binding of the peptide biomolecule to a target to determine the presence of the target in the subject. The presence of the target may indicate a disease in the subject. In other embodiments, the method may include contacting a sample derived from the subject with a peptide biomolecule described herein, determining the level of the target in the sample, and comparing the level of the target in the sample to a control level of the target, where a level of the target different from the control level indicates a disease in the subject. In some embodiments, the disease is selected from cancer, metabolic diseases, autoimmune diseases, cardiovascular diseases, and orthopedic disorders, as detailed above. In some embodiments, the target includes a disease marker or biomarker. In some embodiments, the fusion protein may act as an antibody mimetic for binding to or detecting the target.

[0056] How to determine the presence of a target A method for determining the presence of a target in a sample is provided herein. The method may include contacting the sample with a peptide biomolecule comprising one or more fusion proteins described herein, under conditions that allow a complex to form between the peptide biomolecule and the target in the sample, and detecting the presence of the complex. The presence of the complex may be an indicator of the target in the sample. In some embodiments, the peptide biomolecule is labeled with a reporter for detection. In some embodiments, the sample is obtained from a subject, and the method further includes diagnosing, predicting, or evaluating the effectiveness of a treatment of the subject. When the method includes evaluating the effectiveness of a treatment of the subject, the method may then further include modifying the treatment of the subject as necessary to improve its effectiveness.

[0057] Method for determining the effectiveness of a treatment A method for determining the effectiveness of a treatment in a subject requiring such treatment is provided herein. The method may include: contacting a sample derived from the subject with a peptide biopolymer containing a fusion protein as detailed herein, under conditions that allow a complex to form between the peptide biopolymer and the target in the sample; determining the level of the complex in the sample, the level of the complex being an indicator of the level of the target in the sample; and comparing the level of the target in the sample with a control level of the target, wherein if the level of the target differs from the control level, the treatment is determined to be effective or ineffective in treating the disease.

[0058] The time points may include before the onset of the disease, before the administration of therapy, various time points during the administration of therapy, and after therapy has been administered, or a combination thereof. When a peptide biopolymer containing one or more fusion proteins is administered to a subject, the peptide biopolymer may bind to a target, and the presence of the target indicates the presence of the disease in the subject at various time points. In some embodiments, the target includes a disease marker or biomarker. In some embodiments, the peptide biopolymer may act as an antibody mimetic for binding to and / or detecting the target. Comparison of the binding of the peptide biopolymer to the target at various time points may indicate whether the disease is progressing, whether the disease has advanced, whether the therapy is working to treat or prevent the disease, or a combination thereof. In some embodiments, the control level corresponds to the level in the subject at a time point before or during the period in which the subject begins treatment, and the sample is taken from the subject at a later time point. In some embodiments, the sample is taken from the subject at a time point during the period in which the subject is receiving treatment, and the control level corresponds to a disease-free level or the level at a time point before the period in which the subject begins treatment. In some embodiments, the method further includes modifying the treatment or administering a different treatment to the subject when the treatment is determined to be ineffective in treating the disease.

[0059] It will be apparent to those skilled in the art that appropriate modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope or aspect of any embodiment. The compositions and methods provided are illustrative and are not intended to limit the scope of any particular embodiment. All of the various embodiments, aspects, and options disclosed herein can be combined in any variation or interaction. The scope of the compositions, formulations, methods, and processes described herein includes all actual or possible combinations of the embodiments, aspects, options, examples, and preferred ones described herein. Examples of compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed anywhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation or the total mass of the other components in the formulation is disclosed herein as if they were clearly stated. If the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of the term used herein, the meaning of the term or phrase herein shall prevail. Furthermore, the aforementioned considerations are merely illustrative embodiments. All patents and publications cited herein are incorporated by reference for their specific teaching purposes. Various embodiments and aspects of the present invention described herein are outlined in the following sections.

[0060] Term 1 (X-Z1-X-Z2-Z3-X-Z4-Z3) n (In the formula, X is either proline (P) or glycine (G), and the P:G ratio is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z2 is composed of Asp(D), Arg(R), and Glu(E), where the ratio R:D can be any number, and the ratio D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number; Z4 is a polypeptide having controlled reversible phase separation, comprising 10 or more repeat sequences of amino acid sequences (where Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number). The polypeptide described in item 1, wherein X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. The polypeptide according to item 3, wherein Z1 is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D is not greater than 1:5, and the ratio of K:R can be any number. Item 4: A polypeptide according to any one of items 1 to 3, wherein the phase separation depends on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration. Item 5: A polypeptide according to any one of items 1 to 4, wherein n is between 10 and 200. Item 6 A polypeptide according to any one of items 1 to 5, having a molecular weight of at least 5 kDa to 500 kDa. Item 7 A polypeptide according to any one of items 1 to 6, having a molecular weight of approximately 5 kDa to approximately 100 kDa. Item 8: A polypeptide according to any one of items 1 to 7, wherein the phase separation temperature is 0 to 100°C. Item 9 A polypeptide according to any one of items 1 to 8, wherein the phase separation temperature is 4–25°C, about 25°C, 25–37°C, about 37°C, 35–38°C, or >38°C. Item 10: A polypeptide according to any one of items 1 to 9, wherein the polypeptide comprises a modified amino acid, a reporter protein, or an enzyme.

[0061] Item 11 The array is, (GRGDSPYS) m A polypeptide according to any one of items 1 to 10, comprising (wherein m is between 20 and 80).

[0062] Item 12 Sequence IDs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, A plurality of items described in any one of items 1 to 11, comprising an array selected from 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279, or one or more combinations thereof.

[0063] Item 13 (X-Z1-X-Z2-Z3-X-Z4-Z3) n (In the formula, X is either proline (P) or glycine (G), and the P:G ratio is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z2 is composed of Asp(D), Arg(R), and Glu(E), where the ratio R:D can be any number, and the ratio D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number; A pharmaceutically acceptable composition comprising a polypeptide having controlled reversible phase separation comprising 10 or more repeat sequences of an amino acid sequence (where Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number). The composition according to item 14, wherein X is proline (P) or glycine (G), and the ratio of P:G is 1:3 to 3:1. The composition according to item 15, wherein Z1 is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D not exceeding 1:5, and the ratio of K:R may be any number.

[0064] Item 16 Antimicrobial peptides selected from the following: antibody-binding domains derived from Staphylococcus protein A(ZD) (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelicidine-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177) A composition according to any one of claims 13 to 15, further comprising a binding molecule containing one or more of the following: fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV-binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195). Item 17 A composition according to any one of items 13 to 16, which enhances the bioavailability of the bound molecule compared to the bound molecule in its free form. Item 18 A composition according to any one of items 13 to 17, which enhances the expression of the binding molecule compared to the free form of the binding molecule. Item 19 A composition according to any one of items 13 to 18, which enhances the stability of the binding molecule compared to the free form of the binding molecule. The composition according to item 19, which enhances the stability of the binding molecule during expression in prokaryotes and eukaryotes compared to the free form of the binding molecule. Item 21 The composition according to item 19 or 20, wherein the enhanced stability includes resistance to denaturation during freezing, thawing, or freeze-drying. Item 22 A composition according to any one of items 13 to 21 that modulates an enzymatic function, metabolic function, or physiological function in a cell or organism. The composition according to item 22, wherein the modification reduces the bioavailability of the binding molecule. Item 24 The composition according to item 23, wherein the binding molecule comprises a therapeutic or cytotoxic protein or peptide.

[0065] Article 25 A method for enhancing the bioavailability or stability of a protein, comprising one or more proteins, (X-Z1-X-Z2-Z3-X-Z4-Z3) n (In the formula, X is either proline (P) or glycine (G), and the P:G ratio is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z2 is composed of Asp(D), Arg(R), and Glu(E), where the ratio R:D can be any number, and the ratio D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number; A method comprising creating a polypeptide fusion protein having controlled reversible phase separation, comprising 10 or more repeat sequences of an amino acid sequence in which Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), where the ratio between Y:H:W:F:M:V:I:A:L can be any number. The method according to item 25, wherein X is proline (P) or glycine (G), and the P:G ratio is 1:3 to 3:1. The method according to item 27, wherein Z1 is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D is not greater than 1:5, and the ratio of K:R can be any number.

[0066] Item 28 Antimicrobial peptides selected from the following proteins derived from Staphylococcus protein A(ZD): antibody-binding domain (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelicidine-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP- The method according to any one of items 25 to 27, comprising: 1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domain (Yifan or FMRP (SEQ ID NO: 191)); or one or more AAV-binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195). Item 29 The method according to any one of items 25-28, wherein the enhanced bioavailability of the fusion protein can be used for the isolation or separation of a biological molecule. Item 30 The method according to any one of items 25-26, wherein the biological molecule comprises one or more lipids, cells, proteins, nucleic acids, carbohydrates, or viral particles. Item 31 The method according to item 30, wherein the nucleic acid is single-stranded or double-stranded DNA or RNA. Item 32 The method according to item 30, wherein the virus particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measles virus particle, or a herpesvirus particle. Item 33 The method according to item 30, wherein the protein comprises albumin, a monoclonal IgG antibody, or an Fc fusion antibody. Item 34 The method according to item 29, wherein isolation or separation is achieved via reversible phase separation. [Examples]

[0067] (Example 1) Novel manipulation of intracellular condensates using artificially disordered proteins The inventors took a different, supplementary approach to understand how phase behavior is encoded in polypeptides. Drawing analogy from synthetic polymers exhibiting lower and upper critical dissolution temperature (LCST / UCST) phase behavior, the inventors embarked on identifying the minimum peptide motifs that, when polymerized into polymers consisting of many repeat sequences of peptide motifs, would give rise to LCST or UCST phase behavior by systematically scanning the sequence space of native IDPs. Since the sequence complexity of these repeat polypeptides was greatly reduced compared to native IDPs exhibiting LCST / UST phase behavior, the inventors then rationally modified the amino acid repeat sequence motifs that systematically arose along the sequence. These repeat polypeptides can be rationally designed to exhibit both LCST and UCST phase behavior, and their phase behavior can be systematically regulated by amino acid mutations in the repeat sequence motifs. These artificial polypeptides also exhibit the same fundamental principles of intracellular phase separation as native IDPs.

[0068] Having gained insights from these studies into the factors that lead to phase separation in repeating polypeptides, similar to the naturally occurring membrane-less organelle IDPs, the inventors embarked on creating an artificial IDP (A-IDP) that exhibits phase separation in living cells to impart new functionality to cells. The inventors' design is a sequence (G1-R2-G3-D4-S5-P6-Y7-S8) inspired by Drosophila melanogaster Rec-1 Resilin, which is known to exhibit UCST phase behavior and is chemically similar to IDPs, a key component of membrane-less organelles. XXWe started with (wherein xx is the number of repeat sequences from 20 to 80) (Figure 1A). This sequence exhibits UCST phase behavior, which appears to be much more common among native IDPs than LCST phase behavior, so we precisely selected this sequence. Thus, we used the parent (G1-R2-G3-D4-S5-P6-Y7-S8) XX We created a set of 63 A-IDPs consisting of repeating motif sequences and variants having reasonable amino acid mutations in this motif. We characterized the UCST phase behavior of this set of 63 IDPs so that we can quantify the effects of various amino acid mutations and modifications to the chain structure on isomorphic liquid-to-liquid phase separation.

[0069] Next, the inventors used a subset of A-IDPs derived from this library to manipulate intracellular condensates in living cells. The behavior of these intracellular condensates for A-IDPs proved to be remarkably predictable and regulated, with their cytoplasmic lysis and their interactions with the surrounding environment being dynamically controllable. Leveraging these findings, the inventors created intracellular droplets capable of sequestering enzymes, whose catalytic efficiency within the manipulated condensates can be genetically encoded by regulating the MW of the A-IDPs.

[0070] material and method The pET24+ vector was purchased from Novagen (Madison, WI). The gBlock fragments encoding the target repeat IDP (A-IDP) sequence, superfolder GFP (sfGFP), mRuby3, and pcDNA5 vector primers were purchased from Integrated DNA Technologies (Coralville, IA). Ligation enzymes, restriction enzymes, and DNA ladders were purchased from New England Biolabs (Ipswich, MA). BL21 (DE3) chemically competent Escherichia coli (E. coli) cells were purchased from Bioline (Taunton, MA). All E. coli cultures were grown in Terrific Broth medium purchased from VWR International (Radnor, PA). Kanamycin sulfate was purchased from EMD Millipore (Billerica, MA). Protein expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG) from Gold Biotechnology (St. Louis, MO). All salts, 10 / 40kDa fluorescein-labeled dextran molecules, L-(+)-arabinose, L-rhamnose, and fluorescein di (β-D-galactopyranoside) were purchased from Sigma-Aldrich (St. Louis, MO). 1× phosphate-buffered saline (PBS) tablets (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4 at 25°C) were purchased from EMD Millipore (Billerica, MA). KRX E. coli cell lines endogenously expressing mutated LacZ were purchased from Promega (Madison, WI). NHS ester-reactive fluorophores (NHS-Alexa Fluor® 350 and NHS-Alexa Fluor® 647) were purchased from Life Technologies (Grand Island, NY). DNA extraction kits and DNA gel purification kits were purchased from Qiagen Inc. (Germantown, MD). We purchased the Expi293 eukaryotic expression system for HEK293 expression from Thermo Fischer Scientific (Waltham, MA).We purchased Whatman Anotop sterile syringe filters (0.02 μm) from GE Healthcare Life Sciences (Pittsburgh, PA). We purchased ABIL® EM 90 and TEGOSOFT® DEC surfactants from Evonik Industries (Essen, Germany). We purchased a single emulsion droplet generating tip from Dolomite Microfluidics (Royston, United Kingdom). We purchased a syringe pump from Chemyx Inc. (Stafford, TX).

[0071] Proteomics analysis A literature search revealed that a good list of intrinsically disordered proteins or protein regions exists in genes known to form membraneless organelles. Each gene was divided into disordered and ordered regions according to the predictors of the native disordered region (PONDR) VSL2 algorithm, which are quasi-predictors of protein disorder of various lengths. Amino acid amounts were standardized relative to the total protein length.

[0072] gene synthesis Each octapeptide amino acid motif, inspired by the inventors' proteomics analysis, was amplified 20-fold in silico. This repeat amino acid sequence was incorporated into an algorithm to generate an optimal non-repeat DNA template derived from the repeat protein gene. This 20-mer repeat sequence gene was then ordered from IDT along with a Gibson assembly overhang for easy insertion into the modified pET24+ vector. To increase the total number of repeat sequences in the gene, the inventors performed a repeatable cloning process of recursive directional ligation by plasmid reconstruction, adding an additional 20 repeat sequences during each step. The cells were transformed into the desired E. coli strain, BL21(DE3) for recombinant expression and single-plasmid confocal experiments, and a modified BL21(DE3) cell line, which Promega calls KRX, containing a mutated LacZ gene for enzyme experiments.

[0073] In experiments using dual expression, the gene was inserted into the pBAD33.1 vector by cleaving a custom pET24+ vector and pBAD33.1 cleavage using Hind III and Xba I. The target gene was isolated from the housing pET24+ vector using gel purification and then ligated into a similarly cleaved pBAD33.1 vector. Co-transformation was performed using approximately 1 ng of each plasmid at a final concentration on a kanamycin / chloramphenicol dual-selection plate.

[0074] Protein expression, purification, and characterization Each individual liquid culture of a BL21 Escherichia coli strain possessing the inventors' genes as defined in Table 2 or Table 3 was inoculated from frozen glycerol stock into 5 mL of Terrific Broth (TB) medium and grown overnight (16-18 hours) until confluence. The culture was then inoculated at a 1:200 dilution in 1 L of TB medium supplemented with 45 μg / mL kanamycin. The cells were grown in a shaking incubator (approximately 200 RPM) at 37°C for 9 hours, at which point protein expression was induced by adding 500 μM IPTG (final concentration). The cells were then incubated for a further 18 hours at 37°C (with shaking at approximately 200 RPM). The proteins were then purified from the insoluble cell suspension fraction. In short, the cell pellet was isolated by centrifugation of the culture at 3500 RCF and resuspending in 20 mL of milli-Q water. Next, the cells were lysed by sonicating the cell solution for 2 minutes, pulsed for 10 seconds, and then left to stand on ice for 40 seconds (Misonix; Farmingdale, NY).

[0075] The suspensions of each lysate were centrifuged at 20,000 RCF for 20 minutes to obtain soluble and insoluble fractions. The supernatant was discarded, and the insoluble fraction was resuspended in approximately equal volumes of 8M urea + 150mM PBS (approximately 6-8 mL). For proteins with fluorescent fusion tags, the insoluble fraction was resuspended in 3 × insoluble volume in approximately 2M urea to prevent protein misfolding. This suspension was warmed in a 37°C water bath for 10 minutes, and then centrifuged at 20,000 RCF for 20 minutes. The supernatant was collected from this suspension and dialyzed at 4°C in a 1:200 milli-Q aqueous solution using a 10kDa membrane (SnakeSkin®, Thermo Fischer Scientific). The dialysate was changed twice over a period of 48 hours. Both insoluble and soluble components were collected from the inside of the dialysis bag and centrifuged at 3500 RCF for 10 minutes at 4°C. The supernatant was removed, and the remaining insoluble pellet containing the target protein was freeze-dried for a minimum of 3 days to remove all water from the pellet. Protein purity was characterized by tris-HCl (Biorad, Hercules, CA) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a gradient of 4–20% and staining with either 0.5M copper chloride or SimplyBlue® SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.

[0076] Fabrication of water-in-oil droplets using chip microfluidics To produce water-in-oil emulsion droplets, two liquid phases—an aqueous phase containing the target protein dispersed in 150 mM PBS and an organic continuous phase containing 75% / 5% / 20% volume / volt TEGOSOFT® DEC / ABIL® EM90 / mineral oil—were injected into a microfluidic droplet generator at a constant flow rate using a precise syringe pump. The flow rate of the dispersed continuous fluid was adjusted to ensure droplet formation in the dripping regime, and in these experiments, a constant flow rate of 500 μL / hour for the organic continuous phase and 50–75 μL / hour for the aqueous dispersed phase was achieved. Droplet production in the microfluidic device was monitored using a 5× objective lens on an inverted microscope (Leica) equipped with a digital microscope camera (Lumenera Infinity 3-1 CCD).

[0077] Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy was performed using an Aviv Model 202 instrument and 1 mm quartz cell samples (Hellma). A-IDP was prepared by dissolving the purified and lyophilized product in 5 mM PBS, pH 7.4, at a final concentration of 10 μM. CD spectra were obtained at 50°C, 260 nm to 180 nm, with a 1 nm step and an average time of 0.5 seconds. Data points using dynode voltages above 500 V were ignored in the analysis. The CD spectra were corrected for the 5 mM PBS buffer signal at 50°C. This data collection was repeated three times, and the average of the three measurements was expressed as the molar ellipticity.

[0078] light scattering Dynamic light scattering (DLS) measurements were performed over a temperature range of 10–80°C using a Wyatt DynaPro temperature-controlled microsampler (Wyatt Technology, Santa Barbara, CA). Samples for the DLS system were prepared in 1×PBS and filtered through a 0.02 μm Whatman Anotop sterile syringe filter (GE Healthcare Life Sciences, Pittsburgh, PA) into a 12 μL quartz crystal cuvette (Wyatt Technology, Santa Barbara, CA). Five acquisitions were made for 5 seconds at each temperature, and the results presented represent the average R of the sample at each temperature. h It represents.

[0079] Temperature-controlled UV-Vis spectrophotometry Cloud point transition temperature (T t The saturation point was determined via temperature-controlled spectrophotometric analysis using a Cary 300 (Agilent Technologies). Samples containing various concentrations of protein in 150 mM PBS were cooled at 1°C / min, while the absorbance at λ=350 nm was recorded at 1°C intervals. The absorbance was standardized to the absorbance at the highest temperature point collected, corresponding to the more soluble point during a given experiment. The cloud point was determined as the maximum value in the first derivative of absorbance as a function of temperature. The transition temperature was calculated by the point of the smallest slope. The saturation concentration was defined by a natural logarithmic approximation line constructed from the minimum values ​​of three volume fractions. The error bars are the standard error of the average of three repeats of the minimum values ​​of the three transition temperatures.

[0080] Dextran incorporation experiment Dextran molecule [WT]-20 and [Q 5,8 The ]-20 was incorporated into a phase separation space, and the A-IDP isolates from the surroundings were quantified. Fluorescein isothiocyanate-labeled dextran (10 kDa, 40 kDa, Sigma-Aldrich, St. Louis, MO) were used to isolate unlabeled [WT]-20 and [Q] at final concentrations of 4 mg / mL and 1 mg / mL, respectively, at 60°C. 5,8The sample was added to a 4 mg / mL solution of ]-20. The soluble sample was then transferred to a glass slide at room temperature and covered with a #1.5 coverslip. After incubation below the transition temperature for 1 hour, the sample was imaged using an upright Zeiss Axio Imager D2 microscope equipped with a 20× objective lens and an appropriate filter set (ex470 / 40, em525 / 50). Fluorescence intensity was calculated from the liquid-internal / external background-corrected fluorescence intensity in ImageJ, which was divided using bright-field images of the phase-separated space.

[0081] Sample preparation for temperature gradient experiments A high-concentration A-IDP stock solution (60% by mass) was prepared by resuspending a mass of lyophilized A-IDP pellets in an appropriate volume of phosphate-buffered saline (PBS) at pH 7.0. The concentration was converted to mg / mL, assuming an A-IDP density of 1 g / mL. The RLP stock solution was heated in a water bath at 85°C for 60 minutes, with periodic mixing and sonication to ensure homogeneity. Lower concentration samples were prepared by mixing the initial stock solution with PBS at pH 7 by volume. To prepare temperature gradient microfluidic (TGM) measurements, the solution was added by capillary action to a 12 mm × 1 mm × 0.1 mm rectangular borosilicate glass capillary tube (VitroCom, Inc.), which was then sealed with wax to prevent evaporation and convection of the sample. The capillary tube was maintained at 65°C during the addition process, in contact with an 85°C hot plate housed in an incubator. The high-temperature environment ensured that the RLP solution was maintained above the critical phase transition temperature (approximately 85°C for [WT]-20). Capillary arrays were prepared by taping several capillaries together. Before subjecting the arrays to temperature gradient experiments, they were placed in an oven at 85°C for 10 minutes.

[0082] Measurement of phase transition temperature on a temperature gradient apparatus A temperature gradient apparatus was used to apply a linear temperature gradient to the A-IDP solution. This was achieved by placing a glass capillary array in a thermal environment with one end in contact with a heat source and the other with a cooling sink. The sample was then immersed in white light. This light was scattered by phase-separated A-IDP droplets at a low temperature and imaged via a dark-field microscope. The temperature gradient was adjusted for each experiment using two reference solutions placed alongside the target A-IDP sample. The reference solution for temperature adjustment was 10 mg / mL in H2O with a MW of 1.868 × 10⁻¹⁰. 5 It contained poly(N-isopropylacrylamide) (PNIPAM) (Polymer Source, Inc.) in a concentration of g / mol. High-temperature preparation reference was 10 mg / mL in a 1M NaCl aqueous solution, with MW = 9 × 10⁻⁶. 5 The solution contained poly(ethylene oxide) (PEO) (Sigma-Aldrich) in a concentration of g / mol. LCSTs of each reference solution were obtained using a melting point apparatus that measured light scattering intensity while increasing the temperature at a rate of 0.5 K / min. When placed in a temperature gradient apparatus, the reference solution became turbid at temperatures above the LCST. Pixel positions of the LCST were obtained by comparing the light scattering intensity with a low-intensity baseline on the cold side of the capillary. Assuming a linear relationship between position and temperature, the temperature gradient was calculated using the pixel positions and LCSTs of the two samples.

[0083] Phase diagram binodal approximation The approximate dilution region, overlapping region, and semi-dilution region of the phase diagram obtained by the inventors were calculated using the approximation method for the lower critical solution transfer polypeptide, which was adapted to the upper critical solution transfer polypeptide, as previously described.

[0084] Simply put, for low volume fractions (φ<0.1), A-IDP roughly shows the normal log-reliability at the UCST cloud point for volume fractions observed with other repeat sequence polypeptides. For high-density regimes (φ>approximately 0.4) using the surface tension scaling method already described for elastin-like polypeptides, the inventors have found that [WT]-20 and [Q 5,8The proportionality constant (A) and estimated theta temperature (θ) of ]-20 were determined to be A = -0.00092, θ = 389 K and A = -0.00092, θ = 392 K, respectively. In the poor solvent, the surface tension of the dilute phase droplet γ can be expressed by the formula γ ≈ C kTb² φ²” (where b is the Kuhn length of the polypeptide (b = 2.2 nm as measured by Flügel and collaborators for other repeating polypeptides), and C is an adjustable coefficient). ^ 2 * A * By substituting (T-θ), the inventors have derived the equation for the temperature dependence of the coacervate volume fraction.

number

[0085] In relation to the critical point in the so-called Ginzburg zone, it is necessary to describe the phase behavior of polymer solutions using the critical Ising model. The phase boundary in the critical zone is determined by mean-field theory;

number

[0086] Whole-cell fluorescence intensity measurement Cells were grown overnight in 5 mL of TB medium from a glycerol stock. Total sfGFP fluorescence and OD were recorded in conjunction with fluorescence or confocal imaging. 600 The cells were analyzed. In short, 50 μl of cell cultures from various time points were resuspended in 1 mL of 150 mM PBS. Using a combination of UV-Vis spectrophotometric signals from NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA) and fluorescence spectra from NanoDrop 3300 (Thermo Fisher Scientific, Waltham, MA), the inventors calculated the relative ratio of sfGFP fluorescence normalized to cell density. Using this information in conjunction with image analysis, the inventors were able to determine the intracellular saturation concentration normalized to cell density.

[0087] Temperature-controlled fluorescence microscopy of primitive cell droplets and E. coli bacteria. Water-in-oil droplets were collected on a glass microscope slide and cooled using a precise Peltier heating and cooling stage (Linkam LTS120) equipped with a temperature control unit (Linkam PE95). Alexa Fluor 350 labeling (25% molar fractionation N-terminal labeling) [Q 5,8The spatial distribution of A-20 and Alexa Fluor 594-labeled +4 Net was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective lens and appropriate filter set. Similarly, intracellular patterning of A-IDP-superfolder GFP over time was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective lens and appropriate filter set (e.g., 470 / 40, e.g., 525 / 50). Cellular fluorescence was calculated using ImageJ software. The temperature gradient was started at various temperatures but was always set to a constant rate of 5°C / min.

[0088] Transient transfection of [WT]-20-sfGFP in HEK293 cells [WT]-20-sfGFP was extracted from a pET24(+) vector using polymer chain reaction (PCR). Briefly, forward and reverse primers were resuspended with 1 ng of pET24(+) plasmid containing the [WT]-20-sfGFP gene fusion. The gene was finally constructed using Gibson assembly after 30 cycles of PCR at [98°C, 1 min; 65°C, 30 sec; 72°C, 2 min], followed by gel purification. The pcDNA5 vector containing [WT]-20-sfGFP was transfected into HEK293 cells according to the manufacturer's instructions (Expi293 expression system, Thermo Fischer Scientific, Waltham, MA). On day 5 of transient transfection, cells were spun down at 500 RCF for 10 minutes at room temperature and resuspended in 150 mM PBS for imaging.

[0089] Confocal images of A-IDP-sfGFP fusions regarding spot formation and colocalization. Cells were prepared as follows: Cells were inoculated overnight with selected proteins from a bacterial glycerol stock into tubes containing 5 mL of TB medium. After 16 hours of growth, induction with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, MO) was added to each of the target flasks. Samples were collected at the indicated time points and prepared for imaging as follows: 50 μL of cell suspension was pelleted at room temperature for 1 minute under 20,000 RCF. Cells were then OD-350 with a patch length of 1 cm. 600 The cells were resuspended in a solution of 0.15. 50 μL of the resuspended bacterial cells were transferred to a 384-well plate with a #1.5 glass bottom (Cellvis). A 10-minute equilibration period in the incubation chamber preceded data collection at each time point.

[0090] Images were acquired at different time points using a 63× oil immersion objective lens on a Zeiss 710 inverted confocal microscope equipped with a temperature-controlled incubation (Car Zeiss AG, Oberkochen, Germany). sfGFP fluorescence was detected using a 488 nm excitation laser and a 488 / 594 emission filter. Unless otherwise indicated, data were first acquired at 25°C. All fluorescence quantification and cell portation analysis were performed in ImageJ.

[0091] In the co-existence experiment, cells were grown overnight in a dual antibiotic medium containing 45 μg / mL kanamycin and 25 μg / mL chloramphenicol (final concentration) from a glycerol stock. After 16–18 hours, pET24(+) expression was induced using 1 mM IPTG (final concentration). 24 hours after IPTG induction, the medium was replaced with 5 mL of TB supplemented with 1 mM IPTG and 2% arabinose (final concentration) (Sigma Aldrich, St. Louis, MO). 9 hours after induction using both methods, cells were spun down 50 μL of culture at room temperature and OD with a patch length of 1 cm. 600The solution was resuspended in 150 mM PBS until it reached 0.15. All imaging details were the same, except that mNeonGreen / sfGFP detection was performed using a 488 nm excitation laser and a 488 / 594 emission filter, and mRuby3 detection was performed using a 561 nm excitation laser and a 488 / 561 emission filter.

[0092] Rotating disk confocal imaging of Lac Z alpha-peptide-A-IDP gene fusions for localization and quantification of enzyme activity. Cells were prepared as follows: Cells from a bacterial glycerol stock were inoculated overnight with selected proteins into tubes containing 5 mL of TB medium. After 16 hours of growth, induction with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, MO) was added to each target flask. After approximately 24 hours, 50 μL of cell suspension was pelleted at room temperature for 1 minute under 20,000 RCF. Cells were then divided into OD patches with a 1 cm patch length. 600 The sample was resuspended in 150 mM PBS at 0.15. 50 microliters of the sample was added to a 4-well culture insert (1.5-inch coverslip, Ibidi, Madison, WI) petri dish and incubated at room temperature for 10 minutes. After incubation, 2 μL of 1 mg / mL FDG resuspended in 98% water, 1% DMSO, and 1% EtOH was added. Imaging was started immediately (within 20 seconds), and images were acquired every minute for a total of 30 minutes. Imaging was performed on a Leica DMi8 microscope stand (Oxford Instruments, Abingdon, UK) equipped with a 63× immersion objective lens, and on an Andor Dragonfly Spinning Disk 500 series confocal microscope equipped with a Zyla 4.2 series camera. Converted FDG was detected using a 488 nm excitation laser and a 525 / 50 nm emission filter, and mRuby3 fluorescence was detected using a 561 nm excitation laser and a 600 / 50 nm emission filter.

[0093] K m , V max and kcat Fluorescence spectroscopy to determine Liquid cultures of KRX E. coli containing the target plasmid were grown overnight (16-18 hours) from glycerol stocks. The cells were then induced for 24 hours with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, MO). The cells were pelleted and induced in 140 mM PBS. 600 The substrate was resuspended at approximately 0.15. Various concentrations of FDG were added using a NanoDrop 3300 (Thermo Fisher Scientific, Waltham, MA) while monitoring the fluorescence intensity at 520 nm. The fluorescence intensity of mRuby3 was also calculated using the same instrument as a relative measure of the expression levels of various alpha-peptide fusions. Plots of fluorescence intensities observed at different time points showed the relative levels of substrates (V) at various concentrations. o We obtain alternative measurements of the hydrolysis rate at ). These rates are then converted to a typical Lineweaver-Burk transform, V max and K m This was determined. For consistency of units, [FDG] was set to y=185919 * The fluorescence intensity was converted using the fluorescein standard curve for [FDG in mg] + 1045. This conversion assumes that FDG converted to fluorescein has fluorescence intensity characteristics similar to those of free fluorescein dye.

[0094] Quantitative and statistical analysis of images The following statistical analysis was performed on experiments to determine the intracellular fluorescence intensity of A-IDP-sfGFP at various points after IPTG induction. To determine the intracellular saturation concentration, cell density (OD) was measured for three independent samples. 600 The normalized whole-cell fluorescence was calculated while imaging their intracellular structures. This normalized cell density was recorded as the saturation concentration during the initial observation of phase separation in E. coli, where more than 50% of the cells in the microscope field of view were present. The data were normalized against data collected for [WT]-40 as a reference point. Error bars represent the mean expanded standard error of three separate samples derived from the same original cell suspension.

[0095] Using microscopic images collected at various time points using a confocal microscope, the inventors isolated soluble and spot fractions within cells at different points in time via analysis in ImageJ. Spots consistently generate sufficient pixel density to immerse the detector while simultaneously observing the rest of the cell. Therefore, a threshold of around 2% of the upper limit of total pixel intensity allows for easy separation of this section from the remaining cytoplasm. Using this constant threshold at each time point in each experimental group, the inventors were able to track the overall size of these spots over time with respect to the overall cell size (spots + soluble fraction). The error bars in these data represent the standard error of the mean of the standardized spot (two-phase) range of three images from different fields of view of the sample across the entire cell sample. These two channels are combined and shown separately in Figure 2, but with the same threshold process applied to each image.

[0096] Lacking an automated tool for detecting intracellular phase separation between two images, the inventors manually calculated the intracellular transition temperature. Similar to phase separation detection using UV-Vis spectrophotometry, the intracellular transition temperature was determined as the midpoint between a frame that was reliably homogeneous and a second frame that was reliably biphasic. All transition temperatures were determined in this manner, progressing from solubility to insolubility, regardless of whether the solution was heated or cooled. Due to the subjective level of this evaluation, sample identifiers were concealed from the analysts, and a large number of cells were analyzed in each experiment (n=30). Data were standardized against the first average fluorescence of homogeneous cells at a constant temperature (usually 60°C unless otherwise specified). Error bars indicate the standard error of the mean. The error bars for dextran fluorescence indicate the standard error of the mean fluorescence inside and outside the phase-separated space of three separate fields of view.

[0097] To quantify fluorescein di-β-D-galactopyranoside (FDG) compared to alpha peptides at different expression levels, channels were separated between fluorescence from FDG and mRuby3, respectively. Using the ImageJ particle analysis tool, the green fluorescence range was isolated from the background. If the mean fluorescence of this range was 5% greater than the background fluorescence (the mean fluorescence of the range excluded by the previous particle mask), then the background-subtracted green fluorescence of this particular particle was included in the analysis. These ranges were 0.1 μm. 2 If the value was less than the specified value, the particle was excluded. Using the same particle mask, the average fluorescence of mRuby3 with the background subtracted was calculated for the other fluorescence channel. The inventors report the ratio of these two channels as an alternative to enzyme efficiency. Error bars are the standard error of the mean at each time point.

[0098] To quantify fluorescein di-β-D-galactopyranoside (FDG) inside the cell space versus outside the cell space, the channels were first separated between fluorescence from FDG and mRuby3, respectively. Using the same particle analysis tool in ImageJ, the green fluorescence range was isolated from the background. If the average fluorescence of this range was 5% greater than the background fluorescence (average fluorescence of the range excluded by the previous particle mask), then the background-subtracted green fluorescence of this particular particle was included in the analysis. Their range was 0.1 μm. 2 Particles were excluded if the value was less than the specified value. The ratio of the fluorescence intensity inside the cell to the fluorescence intensity in the extracellular space is the background-corrected average fluorescence of FDG divided by the background fluorescence. Error bars are the standard error of the mean at each time point. To quantify the coexistence, the inventors used the Coloc2 plugin available through the ImageJ software. Using an automatic threshold, the inventors report Mander's coexistence coefficient, which takes into account the intensity for the two channels of the subject already described.

[0099] Identification of the smallest IDP repeat sequences from proteomics analysis and sequence discovery methods. The inventors performed proteomics analysis of 63 IDPs that form membrane-less organelles and investigated their sequence composition. The inventors were particularly interested in categories of amino acids suspected to derive phase behavior through interchain interactions such as charge changes and hydrogen bonding via cation-π and uncharged electrode residues (Figure 1A). The composition of these 63 proteins is remarkably similar to previously identified repeat protein polypeptides exhibiting UCST phase behavior, and their side chain groups are chemically similar to those of synthetic UCST polymers. Using a combination of the inventors' previously developed sequence discovery methods and insights from this proteomics analysis, the inventors designed octa-peptide motifs that are expected to exhibit strong phase behavior when polymerizing to macromolecules under physiologically relevant solution conditions.

[0100] To manage the overwhelming sequence space of all possible mutations in the octapeptide repeat sequence, we classify each amino acid into categories of interchain interactions that may be involved in UCST phase behavior. N, Q, S, and T are classified as polar, uncharged amino acids. RK and DE are a pair of positively and negatively charged amino acids. G and P are placed in a separate category (Figure 3A) considering their unusual structure and importance in promoting the disordered polypeptide backbone. The remaining amino acids are classified as "hydrophobic". To ensure that we modulate UCST phase behavior through mutagenesis of the WT repeat sequence, but not completely eliminate it, we produce only mutations that maintain the type of interaction and merely modulate the strength of the interaction. For example, R and K are both positively charged under normal physiological pH. Therefore, by substituting K for R, we maintain the neutral charge state of the polymer backbone, a parameter known to dramatically affect the observed phase behavior. Similarly, N, Q, S, and T all have the ability to hydrogen bond with water and with each other more readily than aliphatic amino acids such as V. Therefore, by substituting these four amino acids with each other, we maintain an equal number of remaining bonds per chain, each capable of forming a specific type of bond.

[0101] The wild-type (WT) repeating sequence unit is (G1-R2-G3-D4-S5-P6-Y7-S8). 40 (In the formula, 40 refers to the number of repeat sequences). The MW of A-IDP was varied between approximately 15 and approximately 70 kDa by varying the number of repeat sequence motifs from 20 to 80, corresponding to the observed difference in MW in the inherently disordered regions (IDRs) of naturally occurring IDPs (Figure 3B). The parent sequence is referred to as WT in this document, and the inventors use abbreviated notation to refer to sequences throughout the text, with letters in parentheses indicating variants that are substitutions at a specific point. For example, a variant with a complete substitution of Y7 with V in the WT repeat sequence unit results in the notation "[V7]-XX". When a residue is only partially substituted in A-IDP, the inventors use the notation "[BY o :ZV o Using ], the ratio of B to Z represents the ratio of Y to V in the variant, and the subscript o is the position of that residue along the repeat sequence unit. Therefore, for example, [Y7:V7]-40 represents 50% of all Y substituted with V, while [3Y7:V7]-40 represents 25% substitution of Y with V. Double mutants, such as 100% substitution of residues at positions 5 and 8 in the octapeptide repeat sequence, are represented using Q. 5,8 Represented as ]-XX, partial substitutions at these positions using S and Q are represented as [BS 5,8 :ZQ 5,8 It is expressed as ]-XX (wherein B and Z represent the ratio of S to Q). A complete sequence description of the common sequences used throughout this document can be found in Table 1. A complete description of all A-IDP structures, which mix variant and WT repeat sequences along the A-IDP strand, can be found in Tables 2 and 3.

[0102] [Table 2]

[0103] [Table 3] JPEG2026091842000033.jpg159156

[0104] [Table 4] JPEG2026091842000035.jpg222162 JPEG2026091842000036.jpg186162 JPEG2026091842000037.jpg101162

[0105] [Table 5]

[0106] [Table 6]

[0107] A-IDP exhibits robust and reversible UCST phase behavior in an aquatic environment. One advantage of A-IDP is their minimal interaction with other proteins or biomolecules, resulting from their repeating properties. This property of A-IDP, combined with their reversible aqueous two-phase separation, allows for purification without the use of simple columns via UCST phase transition cycling between phase 1 and phase 2 regimes in the phase diagram. An example of this purification process is shown in Figure 1B, where high-expression A-IDP, [Q 5,8]-20, the complete phase can be separated from the soluble fraction of the cell lysate and isolated by centrifugation. Subsequent removal of the low-protein supernatant, lysis of the protein-rich pellet with urea, and dialysis of the soluble fraction in milli-Q water yield 95-99% pure protein, as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figures 1C and 4). The yield of purified A-IDP ranges from 25-300 mg per liter of culture medium in shaker flask cultures.

[0108] A-IDP[WT]-20 and [Q 5,8 ]-20 exhibits UCST phase behavior in vitro. To characterize their phase transition behavior, the inventors utilized three different techniques. First, the inventors utilized droplet microfluidics, forming monodisperse water droplets in oil containing the target A-IDP (Figure 1D). Phase separation can be directly visualized in a spatially restricted compartment of the water-in-oil microdroplet to observe the type of structure formed while cooling the surrounding culture medium. These A-IDPs exhibit classical liquid-to-liquid phase separation, observing multiple nucleation sites of coacervate condensates as they pass through the phase boundary during cooling from 50°C to 10°C (Figure 1D Panel 2). These nucleation sites rapidly coalesce into a single spherical A-IDP dense phase that is moist with each other and equilibrated with a phase with less surrounding A-IDP (Figure 1D Panels 3 and 3-1 / 3-2). Upon reheating to 50°C, the A-IDP-rich phase shrinks in size as the A-IDP redissolves, rapidly re-establishing equilibrium (Figure 1D, panel 4-1 / 4-2). A broader view of this transition can be seen in Figure 5. These data clearly demonstrate that these A-IDPs exhibit reversible UCST phase separation via coalescence and growth dynamics (Figure 1E).

[0109] Secondly, the inventors observed two-phase separation in bulk using temperature-dependent dynamic light scattering (DLS). [Q 5,8A solution of ]-20 was heated to 80°C, and DLS data was collected while the solution was cooled to 10°C. The inventors obtained the hydrodynamic radius (R) of 4 nm as a function of temperature. h We observed a transition from soluble A-IDP molecules (containing ) to aggregates larger than 1 μm (Figures 1F and 6). This transition is very sharp, occurring within a 2°C window at approximately 38°C.

[0110] Thirdly, the inventors characterized UCST phase separation by heating an A-IDP solution at a rate of 1°C / min and cooling it while utilizing temperature-dependent turbidity measurement at a fixed wavelength of 350 nm (Figure 1G). Using this technique, the inventors were able to obtain partial phase diagrams of each A-IDP in question as a function of many different sequence and solution parameters. [Q 5,8 At a dilution volume fraction of ]-40, the inventors have found that the function of the natural logarithm of the A-IDP concentration (T t We observed different UCST cloud points increasing as =m*ln([A-IDP])+b). We also confirmed the complete reversibility of the UCST phase behavior of these A-IDPs, which, after 10 consecutive heating and cooling gradients, the UCST T t This was accompanied by a difference of <1°C (Figure 7).

[0111] The arginine composition, the ratio of aromatics to aliphatic compounds, the charge equilibrium, and the molecular weight define the UCST cloud point. To understand the effect of specific residue substitutions in the octapeptide repeat sequence of A-IDP on phase separation, we prepared a set of “mutant” A-IDPs ranging from 100% a to 100% b (where a is the WT repeat sequence unit). The mixing scheme (periodically inserting the mutant repeat sequence unit b into the WT sequence) is visually illustrated by the color-coded schematic in Figure 8A. The mutant repeat sequences are thoroughly mixed and dispersed along the WT sequence to reduce “block noise” in the copolymer and lead to nanoscale self-assembly in the LCST polypeptide instead of the desired liquid-liquid coacervation. Measurement of the UCST phase behavior of these copolymers is analogous to loss-of-function or gain-of-function screening for the UCST cloud point when motif b is substituted for a (Figure 8A). Due to experimental limitations, loss-of-function phase separation that could not be detected was measured at a volume fraction of 0.1 or less in 140 mM saline aqueous buffer. t <4°C is defined operationally. The total length of the 40 repeat sequences was selected for these A-IDPs to be approximately equal to the median length of the IDR (approximately 320 amino acids) observed in proteomic analysis of naturally occurring IDPs.

[0112] WT and the T of each A-IDP t is a linear function of its volume fraction (φ) (Figures 8B and C). For a given φ, T t is composition (R 2 The function of (=0.97) shows that the behavior of mutant A-IDP, which is a block copolypeptide of a and b, can be linearly described between that of pure polypeptides of a and b. t The linear behavior also allows for the estimation of UCST cloud points for homopolymers exhibiting UCST cloud points beyond the experimentally observable range of detection, thereby enabling each point variation to be placed on a single relative scale (Figure 9).

[0113] The inventors then proposed the following regarding A-IDP, T t Saturation concentration (C) is defined as the concentration at which 37°C is the temperaturesat The effects of 15 different site-targeted substitutions on the above reference WT repeat sequence motif were tested. The inventors determined that a single residue change in the octapeptide repeat sequence was more than two orders of magnitude greater than a certain molecular weight in the range of 1 to 800 μM, and that the percentage change in the amino acid composition of the repeat sequence polypeptide was standardized to the degree of substitution defined by C sat We found that this can be changed (Figures 8D, E, and F). This is preferably about 1 μm and can be visualized by normalizing to the saturation concentration of [WT]-40, shown by the horizontal dotted line in Figure 8F. We found that the changes in the chain structure resulting from these mutations are T t and C sat We do not believe these effects are related to [the underlying cause]. In fact, circular dichroism spectrophotometry shows that mutant A-IDP is structurally disordered, consistent with their G and P-rich composition (Figure 10). These substitutions provide quantitative evidence of the importance of the interaction between R and aromatic residues in the A-IDP repeating sequence motif. When Y is substituted, the inventors substituted W, F, or H from 66°C to 123°C, 59°C, and about 2°C, with φ=10. -3 A dramatic shift in the UCST cloud point is observed (Figure 9A). These data indicate that the interaction between the cationic side chain of R and the aromatic side chains of W, F, Y, and H is a key driving force for phase separation, but the strength of these interactions is side-chain group dependent, in the order W>>>Y>F>>>H. Similarly, by substituting R with K, the UCST cloud point temperature is lowered, and therefore the phase boundary (Figure 8E) is lowered (Figure 8E), and C sat This increases (Figure 8F).

[0114] The inventors then focused on the effect of A-IDP MW on the phase behavior, and since this MW range covers 75% of the IDR in the inventors' proteomics analysis of native IDPs, the inventors selected A-IDPs with MWs between approximately 17 kDa and approximately 70 kDa (Figure 8B). The inventors' results show that MWs have at least as much effect on the UCST cloud point as amino acid substitutions (Figure 11A). The inventors studied the range of approximately 17-70 kDa, T t We observed that the effect of MW on can be approximated using a linear approximation of the natural logarithm of MW (Figure 11B). By simply doubling the MW of [WT]-40, we were able to approximate the C shown by several native IDPs. sat Similarly, prediction of nanomolar concentration regime C sat We were able to produce A-IDP having the following properties (Figure 11C). In particular, by changing both MW and composition, we were able to produce 10 -4 ~10 2 Beyond 7 digits in the μM range, C of A-IDP sat It can be varied.

[0115] T tIn addition to composition, concentration (φ), and MW, several other parameters exist that have a measurable effect on UCST phase behavior but do not eliminate UCST phase behavior under physiologically relevant conditions. Uncharged polar substitution, G / P ratio, repeat sequence polypeptide syntax, solution salt content, pH (in the absence of H), and negatively charged amino acid (E vs. D) identity all bring about smaller changes to the UCST binodal phase boundary than MW, volume fraction, aromatic:aliphatic amino acid ratio, and R content (Figure 9B-C). The N-terminal residue of P6 appears to have a unique impact on the UCST binodal boundary, as compositionally identical A-IDP shifted the UCST binodal line depending on the position of the polar uncharged residue at position 5 of the octapeptide repeat sequence (Figure 12A). The inventors also produced and tested a non-repeating but compositionally identical version of [WT]-20 and observed the minimal effect of scrambling the amino acid sequence on the UCST binodal (Figure 12B). In summary, these results suggest that three parameters, aromatic:aliphatic ratio, volume fraction (φ), and MW, are related to the UCST phase boundary or C in vitro. sat This demonstrates the most important aspect of controlling this.

[0116] A-IDP produces a dense-phase separated condensate at a saturation concentration mediated by the amino acid composition. C in the dilute regime of the UCST phase diagram of A-IDP sat And by observing that the binodal phase boundary can be thoroughly modified by amino acid substitution, the inventors became interested in the factors that modulate the high-concentration regime of the A-IDP phase diagram. Polypeptide [WT]-20 and [Q 5,8-20 is sufficiently abnormally expressed for recombinant proteins, with a yield of about 500 mg / L in shake flask culture, facilitating purification over 1 gram of material to directly measure UCST cloud point behavior (>0.1) at high volume fractions of these A-IDPs. To minimize the amount of material required, these experiments were performed in a multiplexed linear temperature gradient microfluidic device mounted on an upright optical microscope, and the phase separation was determined by the temperature at which it occurred, as visualized by a noticeable increase in light scattering intensity, T t could be quantified. These experiments produced binodal phase boundaries similar to the turbidity measurements typically performed in a UV-vis spectrophotometer (Figure 13), showing that the approximately 25 °C difference between the two binodal lines of [WT]-20 and [Q 5,8 -20 was maintained across the range of volume fractions tested. This corresponds to an increase in the A-IDP volume fraction in the dense phase (φ2) from φ2 = 0.4 for [Q 5,8 -20 to φ2 = 0.55 for [WT]-20 at an isotherm of 37 °C. In addition to the description of these phase diagrams, the inventors did not observe the fluorescence partitioning of dextran into the dense phase, indicating that phase separation in the presence of small (10 kDa) and high (40 kDa) MW fluorescently labeled dextrans shows that both [WT]-20 and [Q 5,8 -20 droplets are highly excluded (Figure 14A - B). These data, combined with the inventors' ability to readily purify A-IDPs from bacterial cell lysates using phase separation, show that A-IDPs form highly excluded droplets in vitro at physiological solutions, temperatures, and pH factors (φ2 > 0.4).

[0117] A-IDP shows the range of T sat controlled as a function of concentration in eukaryotic and prokaryotic cell lines, and C t varies over more than seven digits satUsing this set, the inventors sought to (1) understand the dynamics of droplet assembly in living cells and (2) determine whether it proceeds in vivo as well as in vitro. To explore these two issues, the inventors used 1–815 μM C ​​with either a MW of approximately 17 kDa or approximately 32 kDa. sat A set of IDPs with a range was selected. To visualize the localization of A-IDPs within bacterial cells, each A-IDP was fused to a superfolder version of green fluorescent protein (sfGFP) (Figure 15A).

[0118] Fusion of sfGFP [WT]-20, [WT]-40, [3Y7:V7]-40, and [Y7:V7]-40 to A-IDP did not eliminate the phase behavior but shifted the phase diagram (Figures 15B and 16). Despite this shift, using a confocal fluorescence microscope, we were able to observe the formation of intracellular droplets of [WT]-20-sfGFP in both transfected human embryonic kidney (HEK) cells and Escherichia coli (Figures 15C and 15D, respectively). Interestingly, in the in vitro environment of aqueous droplets in oil, we observed that nucleation occurred at multiple points in the aqueous compartment, but over time, all individual coacervate plaques coalesced into a single, gigantic coacervate plaque. This indicated the absence of a significant energy boundary for dispersion or coalescence. In HEK cells, coacervate plaque nucleation also occurred at multiple locations throughout the cell. However, unlike in vitro conditions, these spots never coalesced into a single coacervate droplet; instead, individual coacervate spots ranging in diameter from 2 to 4 μm were dispersed within the cytoplasm of HEK cells (Figure 17).

[0119] In contrast, phase separation in E. coli is significantly different from that in eukaryotic cells. The initiation of the UCST phase transition in E. coli is similar to that in HEK cells and in vitro, with small, high-density fluorescent spots indicating that the A-IDP concentration in the cells is C satThey form after exceeding a certain threshold, and then their size grows over time (Figure 15G). As more A-IDP is expressed over time, the growth in the size of these spots is due to the absorbance at 600 nm (OD 600 The sfGFP fluorescence measurements from a standardized bulk E. coli population are consistent with those obtained from the same standardized bulk E. coli population. The increase in fluorescence over time indicates that the intracellular concentration of the A-IDP-sfGFP fusion increases with prolonged protein induction time (Figure 18). However, unlike HEK cells, and similar to in vitro experiments, these spots in E. coli coalesce to form a single coacervate droplet per cell (Table 6). This result suggests a difference in the diffusivity of A-IDP between prokaryotic and eukaryotic cytoplasm, suggesting that the boundary for the dispersion and coalescence of coacervate droplets in E. coli is much lower than in HEK cells. At the same time, the remaining dilute regime remains at a relatively constant concentration (Figure 19). Simultaneously, these results suggest that the overall intracellular concentration of A-IDP increases over time, buffering the cytoplasmic concentration of the protein and remaining constant, while the coacervate volume increases in correlation with cell size.

[0120] [Table 7]

[0121] Similar to in vitro studies, MW and aromatic:aliphatic content influence droplet formation in E. coli. Doubling MW from [WT]-20-sfGFP to [WT]-40-sfGFP is sufficient to induce droplet formation, even before A-IDP induction, possibly due to leaky transcriptional regulation. sat This reduces (Figure 15D). Similarly, increasing the aliphatic content using V at the expense of Y reduces C to concentrations that are not measurable over time in these experiments. sat This increases (Figure 15E). This is probably due to the effect of intermolecular crowding within the cell, in vivo C satThe difference is not as dramatic as predicted by in vitro experiments using the A-IDP-sfGFP fusion (Figure 22F), but the inventors used both the MW of A-IDP and the aromatic:aliphatic ratio to adjust intracellular C sat by at least one order of magnitude.

[0122] A-IDP exhibits reversible UCST droplet formation in E. coli By increasing the polypeptide volume fraction, the binodal line can be passed through into the two-phase regime under isothermal conditions. This line can pass at a constant volume fraction by decreasing the quality of the solvent or the chi parameter (χ). Experimentally, this is most easily achieved by lowering the temperature of the bulk solution. Similar to the UCST phase behavior of A-IDP in vitro, A-IDP shows reversible UCST phase separation inside cells, which is reversible by repeating four cooling and heating cycles (Figure 20A). The difference between the cooling transition temperature (T t and C ) and the heating transition temperature (T t and H ) fluctuates by less than 2°C, so the phase separation shows minimal hysteresis (Figure 20B). Interestingly, during multiple heating and cooling cycles, the inventors observed that E. coli showed spatial phase separation memory and the spots formed at the same position as in the first cycle (Figure 2). In addition, the inventors observed that cooling-triggered phase separation resulted in more spots per cell (Figure 21). The larger the number of spots observed using larger MW species, the more spots formed per cell, consistent with previous studies and indicating that it is a function of their dispersion coefficient.

[0123] Increasing the MW of A-IDP increased the T t observed in E. coli (Figure 20C). By manipulating the aromatic:aliphatic ratio while maintaining the MW constant and observing spot formation within individual bacteria at various time points after induction (various intracellular concentrations), the inventors found that T tFurthermore, a partial intracellular phase diagram was obtained as a function of intracellular fluorescence (Figure 20D). This result is important for linking the observed behavior at specific concentrations and coolings for a given construct, while essentially normalizing the cloud point observed for different overall levels of protein expression across the cell population. Again, with increasing concentration, the inventors observed an increase in the UCST cloud point, but the rate of increase with increasing concentration did not appear to follow normal log-confidence.

[0124] Novel design of functional A-IDP droplets in cells To understand the potential of using spatially enclosed intracellular coacervate droplets to perform novel functions, the inventors asked the following questions: (1) Can intracellular coacervate droplets recruit other molecules, and if so, what are the size limits of such molecules?, and (2) Can these molecules interact with A-IDP to impart novel functions to the droplets?

[0125] To answer these questions, the inventors first investigated whether small molecules could diffuse into and react with A-IDP in coacervate droplets located within E. coli cells. The inventors designed and expressed A-IDP-[3Y7:V7]-40-UAA, which has three copies of the azide, a unique double orthogonal reactive group, and its primary amino acid sequence is listed in Table 3. sat After reaching higher intracellular concentrations, the inventors incubated live E. coli with 1 mg / mL dibenzocyclooctin dye (DBCO-Alexa488) for 10 minutes (Figure 22A). After a single washing step to remove excess dye, the inventors observed fluorescent condensates in the cells. These experiments also show that the φ1 fraction, in addition to the condensates, is labeled. These data clearly demonstrate that small molecules can diffuse from the extracellular environment into pre-formed condensates created by A-IDP phase separation and react with A-IDP.

[0126] Next, the inventors questioned whether larger molecules, such as proteins, could also interact with A-IDP spots. To answer this question, the inventors designed droplet capture experiments based on separate green fluorescent proteins (GFP). The inventors first investigated whether two components of separate GFPs could interact with each other to create a functional GFP molecule if one of the components fused with A-IDP. GFP-11-[3Y7:V7]-40-mRuby3 was co-expressed in the presence of GFP-1-10. Here, since the IDP is fused with mRuby3, the A-IDP condensate emits red fluorescence and can be visualized in cells using a fluorescence microscope. The inventors observed fluorescent GFP only within the condensate, as seen by the co-localization of green fluorescence with red fluorescence from the A-IDP condensate, indicating that fragment GFPs bind to each other to produce an intact, functional GFP molecule that emits green fluorescence (Figure 237A). In contrast, in the absence of GFP-1-10 induction, minimal green fluorescence is present within the red fluorescence condensate (Figure 237B).

[0127] These results indicate that despite steric hindrance imposed by A-IDP and a fluorescent reporter fused to one protein fragment, two GFP protein fragments can find and bind to each other within the cell. However, the intracellular concentration of GFP-11-[3Y7:V7]-40-mRuby3 is low. sat It was not found that, before the phase separation that occurs when the A-IDP condensate is exceeded, the protein partner from previous experiments can be co-expressed and bound in the cytoplasm, and therefore, after the A-IDP condensate is formed, the protein can be recruited. To directly answer this question, we co-transformed E. coli using two plasmids: a Lac operon-controlled plasmid encoding one fragment of GFP (GFP-11) fused to [3Y7:V7]-40, and a second plasmid controlled by the araBAD operon encoding the other fragment of GFP (GFP-1-10). Expression of GFP-11-[3Y7:V7]-40 at 37°C, its intracellular concentration, and its Csat As the process progressed to a higher, sufficiently long duration, the inventors removed the IPTG induction medium and replaced it with an arabinose-containing medium that induces the expression of a larger GFP fragment (GFP-1-10). After arabinose induction, the inventors observed that both the φ1 and φ2 fractions of E. coli contained fluorescently active GFP (Figure 22B). This result suggested that the larger GFP fragment has the ability to permeate pre-formed condensates in cells, find its binding partner despite fusion to A-IDP, and form a complete, functional molecule. Once the complete, functional GFP molecule is recruited into an intracellular droplet, it is then possible to dramatically modify the intracellular solubility of the reconstituted GFP-A-IDP by altering the bulk temperature (Figure 24).

[0128] These experiments clearly demonstrate that small molecules and proteins can be recruited into intracellular coacervate droplets in E. coli, and that proteins can be reconstituted within these droplets. These results suggest a pathway for the novel design of intracellular coacervate droplets using novel enzymatic functions. One of the reasons for proposing the revolutionary development of biomolecular condensates is to regulate the dynamics of various biological functions, including enzymatic reactions; therefore, the inventors selected biocatalysis as the target function. However, there is little experimental evidence to show how enzyme function can be regulated by phase separation.

[0129] To investigate this, the inventors created an A-IDP fusion in which the enzyme can be recruited into an intracellular droplet and its catalytic activity can be regulated. The inventors selected β-galactosidase for two reasons: (1) it has a wide range of small molecule substrates, one of which, fluorescein diβ-galactopyranoside (FDG), is colorless but emits green fluorescence when cleaved by β-galactosidase. Therefore, using a combination of a red fluorescent protein tagged with the inventors' enzyme-A-IDP fusion and fluorescein fluorescence, the inventors can track the coexistence of the enzymatic reaction with A-IDP in real time. (2) The inventors had concerns that the large enzyme fused to the large A-IDP would not be expressed at sufficiently high concentrations in E. coli and therefore would not undergo phase separation in vivo. To mitigate this concern, the inventors utilized a widely used β-galactosidase (LacZ) blue-white screening system in which an alpha-peptide (αp) complements the mutated enzyme LacZΔM15 to produce a functional β-galactosidase enzyme. In the inventors' system, αp is fused to the A-IDP-mRuby3 construct such that the enzyme activity physically binds to A-IDP and subsequently to red fluorescence.

[0130] Our research using DBCO-Alexa488 and separate GFPs laid the foundation for this more complex experiment. DBCO-Alexa488 experiments suggested that small molecules, such as enzyme substrates, could pass through the spots even if delivered extracellularly (Figure 22A). Separate GFP experiments suggested that relatively large proteins could be recruited to the A-IDP condensate to form functional proteins, suggesting that the same should be possible using separate β-galactosidase systems (Figure 22B). Because there are numerous separate enzyme systems or small protein motifs engineered to bind to various intracellular targets, this peptide binding system also represents a more eccentrically located engineered spot platform.

[0131] Accordingly, the inventors fused an α-peptide (αp) derived from LacZ β-galactosidase to the A-IDP-mRuby3 construct. The inventors hypothesized that the αp-A-IDP-mRuby3 protein could bind to other fragments of the enzyme (LacZΔM15 with α-peptide deletion) endogenously expressed in genetically modified E. coli (KRX, Promega) and be recruited into intracellular droplets. After protein induction and the formation of the resulting condensate, the inventors delivered the substrate fluorescein diβ-galactopyranoside (FDG) to the cell culture medium, where it was transported into the cell, hydrolyzed to fluorescein at the active β-galactosidase site, and finally excreted outside the cell (Figure 22C). By tracking the generation of green fluorescence of fluorescein using a confocal microscope, the inventors could specifically observe where and when enzyme activity occurs within the cell and quantitatively track the enzyme activity.

[0132] In our control experiment with αp-mRuby3, we observed the limits of intracellular fluorescence persistence. Since α-peptide itself is known to form inclusion bodies, it is important to note that even in this control experiment, we observed several spots within the bacterial cells. However, upon fusion with [WT]-20, we observed that the fluorescence localized for a sufficiently long time with the A-IDP condensate, which should be observed using a confocal microscope (Figure 22D). Despite this increased coexistence, the total fluorescence production over time was not statistically significant compared to the αp-mRuby3 control (Figure 22E).

[0133] The inventors have increased the MW of A-IDP, and therefore, C satWhen the amount of MW was reduced, the inventors observed the dose-response effect in total FDG fluorescence intensity and coexistence with the αp-A-IDP-mRuby3 fusion (Figure 22D). αp-[WT]-40-mRuby3 and αp-[WT]-80-mRuby3 had 2.5× and 7.5× more converted FDG at 20 mins compared to the αp-mRuby3 control (Figures 22D and 25). Quantification of green and red fluorescence coexistence using Mander's overlap coefficient showed increased coexistence when the α-peptide fused with A-IDP compared to the fluorescence reporter alone (Figure 26). To quantify the observed coexistence, the inventors analyzed individual cells within image frames using green fluorescence that was above the background threshold at each time point. The larger the MW A-IDP, the stronger the intracellular fluorescence, normalized to the background at each time point (Figure 22F). This volume-response effect of MW highlights the mechanism of extended persistence of substrate molecules within the droplet, leading to more efficient green fluorescence conversion.

[0134] In vitro quantification of fluorescence production at various substrate concentrations suggests that this mechanism of action involves the enzyme's catalytic constant (K) increasing as MW increases. cat This suggests a statistically significant increase in ). This constant can be interpreted as the enzyme's "turnover efficiency" or the number of catalytic events occurring per unit time. The inventors compared αp-mRuby3 control with αp-[WT]-20-mRuby3, αp-[WT]-40-mRuby3, and αp-[WT]-80-mRuby3, and K cat We observed increases of 1.4×, 1.6×, and 4.2× in the presence of the product (fluorescein) and labeled A-IDP as a function of MW. Considering our previous observations of the increase in fluorescence in the presence of the product (fluorescein) and labeled A-IDP as a function of MW, we believe that the observed increase in fluorescence corresponds to a higher measurement K. cat We propose that this is caused by an increase in the coexistence of enzyme and substrate in the condensate. The inventors describe the affinity of the enzyme for the substrate using the Michaelis-Menten constant (K m No significant changes were observed in ), suggesting that the A-IDP fusion does not alter the binding constant of the enzyme-substrate complex.cat and K m Using this, the inventors can define catalytic efficiency, which also supports the inventors' hypothesis that the enzyme efficiency in the condensate increases with increasing MW of A-IDP. This enhancement in enzyme efficiency is due to K cat This refers to the degree of change observed by various protein manipulation techniques initially used to improve the product.

[0135] [Table 8]

[0136] The inventors also fused LacZ alpha peptide to A-IDPs with different levels of aromatic content at a given MW (Figure 28A). The inventors hypothesized that the different levels of aromatic content would affect FDG uptake into droplets and therefore affect the overall enzyme activity. Surprisingly, the inventors observed similar overall levels of fluorescence among αp-[WT]-40-mRuby3, αp-[3Y7:V7]-40-mRuby3, and αp-[Y7:V7]-40-mRuby3. However, the dynamics of enzyme activity differed from A-IDPs with higher aliphatic content, which allowed for faster uptake into condensates (Figure 28B). The difference in the ratio of intracellular and extracellular FDG fluorescence between αp-A-IDP-mRuby3 fusions with different aliphatic content was not significant, indicating that MW is the primary driving force for fluorescein and / or FDG persistence within intracellular droplets (Figure 28C). Complete deletion of aromatic residues from the A-IDP repeat sequence unit results in a soluble enzyme that does not form intracellular condensates and whose activity is lower than that of the enzyme formed by the complementarity of LacZΔM15 with an αp-mRuby3 fusion without the A-IDP tag (Figure 29A-B).

[0137] The inventors show herein that A-IDPs, consisting of repetitive arrays of octapeptide motifs inspired by native IDPs, exhibit reversible UCST phase separation in aqueous solution. Despite the simplicity of their sequences, they recapitulate many of the properties seen in more complex native IDPs. The formation and dynamics of their phase separation into coacervate droplets are controlled by two simple design parameters that are genetically encodable at the sequence level, the MW of the A-IDP and the ratio of aromatic:aliphatic residues in the octapeptide repeat sequence. Using these two parameters, the ratio of aromatic:aliphatic and MW, the inventors were able to generate A-IDPs with C sat in the concentration range from nanomolar to millimolar. This work supports the growing evidence for R-aromatic interactions leading to phase behavior and adds further evidence for the molecular hierarchy that exists between the aromatic groups W, Y, F, and H in the tuning of UCST phase behavior. The IDP literature often ignores the importance of MW, but the inventors' results suggest that MW can be more important than composition for the determination of the UCST binodal. The inventors predict that these results will account for strategies to mutagenize native IDPs and design new IDPs and will shift dramatically.

[0138] These design parameters translate accurately from the in vitro to the intracellular environment. The A-IDP phases separate inside the cell by the same principle that brings about their UCST phase separation in vitro, indicating that the same thermodynamic driving forces incorporated into the sequence and molecular weight also regulate the droplet formation dynamics in isolation. Due to the simplicity of their design, the in vivo A-IDP behavior as their phase diagram in vitro, for increasing intracellular concentrations of C sat suggests that the small phases separating the droplets are formed at individual points in a space where their size continues to grow as the overall A-IDP concentration inside the cell increases. This predictable finding is theorized by previous studies but has not yet been concluded.

[0139] Ultimately, these proteins can be used for the novel design of functional intracellular droplets. The inventors rationally designed intracellular splatters capable of binding to and recruiting β-galactosidase-deficient mutants, which are complexes that have not evolved to form intracellular condensates, and can modify the catalytic efficiency of the enzyme-substrate complex. The catalytic efficiency of the reconstituted enzyme in phase-separated coacervate droplets is MW-dependent and increases with the MW of A-IDP. The larger the MW of A-IDP, the more efficiently the substrate is released in the enzymatically active intracellular phase-separated splatters, and K cat As measured by this method, it results in higher catalytic efficiency. These proof-of-concept experiments demonstrate that intracellular droplets can be manipulated to have noncritical functions in living cells, providing a novel platform for intracellular material manipulation. In summary, a wide range of C sat For this study, we synthesized over 60 IDPs, recruited proteins into coacervate droplets within cells, and used proof-of-concept experiments to modulate protein function. These studies form the basis for the novel design of functional intracellular condensates. We anticipate that these A-IDPs will be useful as building blocks that can encode novel functions of intracellular droplets, further studying the functional advantages of phase separation in living cells by constructing novel biological condensates with rapid behavior within living cells. We also predict that these IDPs will prove useful in other biomedical applications beyond the design of intracellular droplets, benefiting from their tunable UCST phase behavior. This connection between the biophysical characterization of half-cellular materials in soft materials science remains an exciting space for manipulating cells and novel biomaterials with novel or improved functions.

[0140] (Example 2) Figures 30A - D show examples of various fusion proteins that are expressed at low levels in prokaryotic expression systems that rescue expression levels when fused to disordered biopolymers, and the use of the phase separation behavior of biopolymers enables recovery into the soluble fraction. This can be done using an mAb-binding protein having a nanobody folding structure that binds to mAb(ZD); a fluorescent fusion protein having a beta-barrel structure (sfGFP); a therapeutic protein peptide (GLP-1) having a strong alpha-helix tendency, an RNA-binding protein (PUMHD) having a tandem repeat structure, and an antimicrobial peptide showing cytotoxic tendency in Escherichia coli.

[0141] Table 8 shows the expression levels of various fusion proteins.

Table 9

[0142] Figure 31 shows the incubation of an mAb with a phase-separating biopolymer fused to a domain derived from protein A that binds to the mAb. First, the biopolymer is bound to the mAb and centrifuged to capture the mAb heavy chain (HC) and light chain (LC). Next, the supernatant is removed, and the pellet is resuspended in an elution buffer that is low in pH and causes dissociation between the biopolymer-ZD fusion and the mAb. The solution is spun down again to obtain an elution supernatant containing pure mAb HC and LC with little other protein contamination. The elution pellet contained the biopolymer and no mAb.

[0143] [[ID=二十]]Figure 32 shows a phase-separated protein ((GRGDQPYQ) fused to the Z-domain of protein A(ZD) 40Microscopic images of a fluorescently labeled mAb (red / white on a grayscale) visualized in the presence of SEQ ID NO: 3) with m=40 are shown. The first frame shows the coexistence of droplets with the fluorescence signal. When the buffer pH drops at t=0, a reversal of the fluorescence signal is observed, suggesting that the mAb completely dissociated from the phase-separated protein-ZD fusion protein (white arrow) and entered the surrounding solution (red). Since the droplet fusion occurs in 60-240 seconds, these biomolecules-fusion proteins retain their liquid-like behavior.

[0144] Figure 33 shows fusion proteins containing various AMPs fused to biomolecules. When the fusion protein is not expressed, cell growth is impaired. 600 The study proceeds using a standard measured by gradually increasing absorbance. When AMP alone is expressed, cell growth halts. When an AMP-biomolecular fusion protein is expressed, standard growth is restored, suggesting a reduction in AMP availability.

[0145] (Example 3) Effect of repeat polypeptide design on in vivo release of glucagon-like peptide 1 (GLP-1) from subcutaneous depots The inventors utilized the phase behavior of chemically inspired repeat sequence polypeptides derived from naturally occurring IDPs to control the bioavailability of resources when occluded by a lipid bilayer in bacteria, and to control micelle assembly when sterically occluded by relatively hydrophilic ELP or RLP molecules exhibiting surfactant-like activity. The inventors were motivated to test the effectiveness of controlling bioavailability in systems bound to an infinite sink, where the dilute phase is in equilibrium with a biological system capable of protein clearance. The Chilkoti lab has extensive experience with subcutaneous delivery of peptide molecules in vertebrates in this precise setup, primarily using LCST polypeptides, with a main focus on the delivery of therapeutic molecules for diabetes and various cancers. Peptide delivery remains a significant challenge in drug delivery. Despite improvements in protein manipulation focused on reducing half-lives, their effective windows, ranging from minutes to hours, make them unsuitable for therapeutic use. Interestingly, while peptides are utilized in various biological applications in nature, the regulation of their activity is often strictly controlled by cell populations capable of responding to phenotypic changes. Therefore, human-made peptide drugs require delivery solutions, and improving the pharmacokinetics of these valuable macromolecules can be beneficial.

[0146] The most common approaches to improving the half-life of peptides include protein manipulation, formulation modifications to extend release, and / or reduction of renal clearance. Sequence manipulation, such as the incorporation of D-amino acids or other chemically challenging amino acid derivatives, limits the proteolytic degradation of proteins but can severely restrict manufacturing options. Encapsulation methods require harsh production conditions, resulting in inconsistent effects on bioavailability or limiting the types of peptide drugs that can be delivered via these methods. Strategies to reduce renal clearance focus on reducing opsonization, including binding to synthetic or biological polymers that increase molecular size and extend half-life, fusing to large proteins, and binding of chemical moieties that allow peptides to piggyback to endogenous biomolecules with slow turnover rates, such as albumin or antibody fragments. These strategies are not without limitations, as they dramatically reduce potency and depend on patient populations consistently expressing these piggyback biomarkers across individuals.

[0147] These engineered polypeptides offer a remarkable solution. Through their primary amino acid sequence and molecular weight, these engineered polypeptides effectively control drug bioavailability by controlling the density and local dilute phases of the bioactive molecule. Unless proteolytic enzymes possess the inherent ability to diffuse polypeptides into the dense phase of the peptide drug depot, the availability of peptide drugs is comprehensively mediated by the polypeptide's primary amino acid sequence. This local dilute phase can then diffuse from the subcutaneous space into circulation and exert its therapeutic effect. Using the polypeptide design principles described herein, the inventors can rationally design drug-release depots that can extend the in vivo half-life of peptide drugs.

[0148] For this study, the relevant peptide drug is GLP-1, a 31-amino acid peptide produced in intestinal L cells that has the ability to exert glycemic control over a wide therapeutic window. Previous experience with GLP-1 polypeptide depots has provided sufficient insight into the design of subcutaneous depots for drug release. (1) Polymeric fusions such as polypeptide ELP reduce the potency of the GLP-1 molecule to approximately 1 / 30th, but this does not interfere with in vivo activity. (2) Zero-order release can be achieved for up to 10 days in mice and up to 17 days in monkeys under optimal conditions. (3) Optimal conditions are an injectable transition temperature of 5-7°C below animal body temperature and a molecular weight of 35 kDa or greater to avoid renal clearance. This optimal 5-7°C below body temperature corresponds to a dilute phase concentration of approximately 1-100 μM, and suboptimal depots are above and below this optimal range. sat This was shown.

[0149] The peptide drug chosen for these experiments is GLP-1 for several reasons: (1) GLP-1 can rapidly exert its therapeutic effect in vivo. (2) GLP-1 is a leading candidate for improved pharmacokinetics with an in vivo half-life of approximately 5 minutes. (3) GLP-1 can be easily studied in an established mouse model of diet-induced obesity, where a high-fat diet increases blood glucose. (4) GLP-1 is a stable peptide drug that eliminates gene fusion to various polypeptide partners, associated confounding variables, and countless delivery strategies. Previous studies have suggested that the transition temperature at injection concentration is a crucial parameter for determining efficacy. However, this misjudges the critical isotherm on the phase diagram as room temperature instead of the operating temperature of the depot, which is defined by the animal's resting body temperature. In a subcutaneous mouse model, this temperature is approximately 35°C. Therefore, we are seeking proteins with different molecular weights and dilute phase concentrations (similar to saturation concentrations at 35°C) at a 35°C isotherm to test in order to observe how these two variables affect the bioavailability of GLP-1. A potential confounding effect in this study is the delivery method. Using polypeptides exhibiting UCST, compared to previously tested C using LCST polypeptides. sat C is evenly distributed within the range sat To achieve this, the cloud point of polypeptide solutions is often dramatically higher than the body temperature of animals. Therefore, one of the inventors' first tests was the establishment and formation of a depot using a soluble small molecule (urea) that can diffuse more rapidly from the injection site than the polymer, thus allowing the polypeptide to be injected in a soluble solvent that rapidly exchanges with the environment to become a poor solvent (Figure 34). Secondly, the inventors physically embedded a dry depot of the shape, size, and volume already described, which rehydrates and begins to release the active polypeptide after an initial delay (Figure 34). The inventors investigated constant doses that rapidly produce depots of similar size in the subcutaneous space.

[0150] Next, the inventors of the present invention have found different C satWe designed polypeptides with similar molecular weights that exhibit the same properties. The inventors achieved this feat by utilizing the aromatic:aliphatic ratio to rationally adjust the binodal properties of the GLP-1 polypeptide fusion, which are the same parameters as before. This allows the inventors to control the therapeutic efficacy of the fusion. sat It became possible to observe the effect of C. sat This is the only parameter that affects bioavailability. The overall size of the molecule is also important for dispersion / convection in the subcutaneous space. Therefore, C in a similar range sat Using this, the inventors test the depot for delivery to increase the overall molecular weight of the molecule. Previous studies by previous lab members have shown that the reduction returns the molecular weight to over approximately 35 kDa.

[0151] gene synthesis Each octapeptide amino acid motif having a desired saturation concentration was fused to the C-terminus of GLP-1. To increase the total number of repeat sequences in the gene, the inventors performed a repeatable cloning step of recursive directional ligation by plasmid reconstruction, adding 20 additional repeat sequences during each step. For recombinant expression, the cells were transformed into the desired E. coli cell line, BL21(DE3).

[0152] Protein purification Each individual liquid culture of a BL21 Escherichia coli strain containing the inventors' gene was inoculated from frozen glycerol stock into 5 mL of Terrific Broth (TB) medium and grown overnight (16-18 hours) until confluence. The cultures were then inoculated at a 1:200 dilution in 1 L of TB medium supplemented with 45 μg / mL kanamycin. The cells were grown in a shaking incubator (approximately 200 RPM) at 37°C for 9 hours, at which point protein expression was induced by adding 500 μM isopropyl-β-D-thiogalactoside (IPTG). The cells were then incubated for a further 18 hours at 37°C (with shaking at approximately 200 RPM). The proteins were then purified from the insoluble cell suspension fraction. In short, the cell pellet was isolated by centrifugation of the culture at 3500 RCF and resuspending in 20 mL of milli-Q water. Next, the cells were lysed by sonicating the cell solution for 2 minutes, pulsed for 10 seconds, and then left to stand on ice for 40 seconds (Misonix; Farmingdale, NY).

[0153] The suspensions of each dissolved substance were centrifuged at 20,000 RCF for 20 minutes to obtain soluble and insoluble fractions. The supernatant was discarded, and the insoluble fraction was resuspended in approximately equal volumes of 8M urea + 140mM PBS (approximately 6-8 mL). This suspension was warmed in a 37°C water bath for 10 minutes, and then centrifuged at 20,000 RCF for 20 minutes. The supernatant was collected from this suspension and dialyzed at 4°C in a 1:200 milli-Q aqueous solution using a 10kDa membrane (SnakeSkin®, Thermo Fischer Scientific). The dialysate was changed twice over a period of 48 hours. Both the insoluble and soluble components were collected from the inside of the dialysis bag and centrifuged at 3500 RCF for 10 minutes at 4°C. The supernatant was removed, and the remaining insoluble pellets containing the target protein were freeze-dried for a minimum of three days to remove all water from the pellets. Protein purity was characterized by tris-HCl (Biorad, Hercules, CA) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a gradient of 4–20% and staining with either 0.5M copper chloride or SimplyBlue® SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.

[0154] Temperature-dependent UV-vis spectrophotometry The turbidity characteristics were obtained for each structure by recording the optimal density as a function of temperature (gradient of 1°C / min) on a temperature-controlled UV-vis spectrophotometer (Cary 300 Bio; Varian Instruments; Palo Alto, CA). t This point was defined as the inflection point of the turbidity characteristics. The samples were measured in 10 μM PBS. Livestock All experimental procedures were performed under protocol A053-15-02, approved by the Duke Institutional Animal Care and Use Committee (IACUC). Six-week-old male C57Bl / 6J mice were purchased from Jackson Labs (strain 000664), grouped in a room with a controlled photoperiod (12-hour light / 12-hour dark cycle), and allowed to acclimate to the facility for at least one week before the start of the procedure. The animals had unrestricted access to water and food and were observed daily for any signs and symptoms of concern. Upon arrival at the facility, the mice were maintained on a high-fat diet (60 kcal% fat) to achieve a diet-induced obesity (DIO) phenotype.

[0155] Endotoxin removal The construct was endotoxin-purified prior to injection by passing the solution through a sterile 0.22 μm Acrodisc filter consisting of a positively charged and hydrophilic Mustang® E membrane (Pall Corporation). The construct was filtered at 37 °C in 2 M urea + 140 mM PBS and then dialyzed against milli-Q H2O at 4 °C, with water being exchanged three separate times over a 72-hour period. Aggregated material was removed from the dialysis bag and pelleted using centrifugation (4 °C, 3500 rpm). Samples were frozen and lyophilized for a minimum of 48 hours.

[0156] Methods for establishing GLP-1 releasing subcutaneous depots In one method, the polypeptide is resuspended at 175 μM in 2 M urea + 140 mM PBS. A total volume of 200 μL is injected into the right posterior flank after shaving and removing all hair using chemical lysis at the injection site. Mice were weighed to determine the injection volume required for approximately 2100 nanomoles of GLP-1 per kilogram of animal body weight. The injection dose did not exceed 200 μL. In a second method, animals were anesthetized with isoflurane and then a small incision was made in the right posterior flank using surgical scissors. The incision site was pre-sterilized according to Duke Animal Care guidelines. Next, a pre-weighed dehydrated polypeptide pellet was inserted subcutaneously. The pellet rapidly rehydrated and adhered to the skin tissue, thereby sealing the incision site and closing the skin flap using only a small amount of surgical adhesive.

[0157] Blood glucose measurement and mass measurement Mice were placed in clear stationary tubes. Their tails were wiped with 50% ethanol in sterile water and then dried. A small lancet was used to make a small incision adjacent to the tail vein. The first droplet of blood was blotted. Blood glucose was quantified by applying the second droplet of blood to the test strip of an AlphaTRAK2 blood glucose meter (Abbott Laboratories). Mass was measured on a scale zeroed using a container in which the mice were placed for a short time.

[0158] Statistical analysis The number of experiments for both in vitro and in vivo studies was selected based on knowledge gathered from previous experiments or other publicly available data. Due to the small sample size (n ≤ 6), group normality was not examined. Differences between groups were similar, except for the untreated vs. in vivo treated group. This is not unexpected, given the lack of glucose control in the mouse models tested. Blood glucose and percentage changes in mass studies were analyzed using repeated-sequence ANOVA, followed by lower-limit ANOVA and Dunnett's test for multiple comparisons. A two-tailed Student's t-test was used to compare two groups. Blinding was not performed. Analysis and data processing were performed using Igor and R software.

[0159] result The inventors faced two strategies involving the possibility of subcutaneously establishing a depot for UCST polypeptides having a transition temperature far above the safe biological temperature range, thereby preventing solubility to insoluble transition during cooling to body temperature. The two strategies are 1) using urea to lower the solution cloud point for injection, and 2) injecting a concentrated, dehydrated GLP-1-IDP fusion, thereby releasing the peptide fusion. The inventors decided to directly visualize this effect via fluorescence tomography. The inventors selected (Gly-Arg-Gly-Asp-Ser-Pro-Tyr-Gln)-40, a model IDP with a predicted transition temperature of >70°C at the required injection concentration (175 μM or 1.2 mg, corresponding to an equal dose of 1000 nmole / kg of the GLP-1-ELP fusion used in previous studies). Using a near-infrared fluorescent tag (CW800) bound via NHS-ester chemistry with a free amine, the inventors visualized the localization of polypeptides in the posterior flank.

[0160] The inventors have found that the inclusion of polypeptides in 1 M urea in solution is turbidity observed up to approximately 25°C. Therefore, by resuspending the model polypeptide in 2 M urea + PBS at 175 μM, the inventors can inject it in a soluble state under ambient conditions. Considering the two-order-of-magnitude difference in molecular weight between urea and the model polypeptide, the inventors hypothesize that urea rapidly diffuses into the subcutaneous space, thereby freeing the remaining polypeptide in the poor solvent, which in turn transfers to Insights. During injection, the inventors observed something resembling a "burst" release of polypeptides (Figure 35). This was characterized by a giant fluorescence area at the center of the mass along the axis of the needle's injection path. After the first 8 hours, the intensity of the center of this mass decreased, slowly reaching an equilibrium state over the first 2 days. The center of this mass disappeared by day 14.

[0161] The inventors also injected a dehydrated coacervate having the same total protein content as in the urea experiment. Here, the inventors observed completely different behavior. The inventors' initial intention was to use convection from the syringe to push the dehydrated depot from the needle point into the subcutaneous space upon contact with water, so that the dehydrated depot would firmly bind to the hydrophobic needle (Figure 36). Thus, the inventors implanted the material using forceps, thereby placing the dehydrated pellet under the skin through a small incision. During implantation, the inventors observed a very small area of ​​fluorescence that slowly expanded over the first 90 minutes. The center of the graft mass did not move noticeably over the course of two weeks. However, the depot mass decreased over time, mainly slowly releasing material around the depot during the first three days after implantation. Comparing these two injection strategies, the inventors decided to proceed with the dehydration depot strategy due to the lack of burst release and increased depot duration.

[0162] Fusion of GLP-1 to the N-terminus of polypeptides was generally well tolerated. The inventors observed minimal yield loss from recombinant expression, with most constructs expressed at 25–50 mg / L. As previously stated, the inventors used a general range of C of approximately 0.1, 10, and >100 μM, corresponding to slow release, optimal release, and nearly soluble release from the depot. sat We wanted to design peptide-polypeptide fusions having [3Y:V]-20, [Y:V]-20, and [3V:Y]-20. sat This roughly corresponds to [3V:Y]-20. It was expected that it would not exhibit phase behavior under physiological conditions, and therefore, six His residues were fused to the C-terminus of the polypeptide, and it was purified from the soluble fraction using chromatography.

[0163] The phase behavior of these polypeptide fusions was measured as before using temperature-dependent UV-vis spectrophotometrics. In determining the UCST binodal lines, the inventors used approximately 30 μM of C sat GLP-1-[3Y:V]-20 exhibits this property, and approximately 500 μM of C sat We identified these two proteins that actually exhibit the desired phase behavior using GLP-1-[Y:V]-20 (Figure 37). These roughly correspond to the values ​​predicted by single-selection RIDP. The predicted GLP-1-[3V:Y]-20-His6X did not exhibit phase behavior under physiological conditions. After endotoxin purification, 1.2 mg each of GLP-1-[3Y:V]-20, GLP-1-[Y:V]-20, and GLP-1-[3V:Y]-20-His6X were weighed and implanted into the hind flanks of C57Bl / 6J mice fed a 60% fat diet. In addition to these three groups, there was another group that received saline injections. Throughout the study, the inventors measured blood glucose levels via tail vein blood collected at 0, 1, 2, 4, 8, and 24 hours, and then at a total of 8 days later.

[0164] Blood glucose data can be visualized in Figure 38. Overall, our strategy of implanting dehydrated depots was successful in controlling blood glucose. We are also keen to observe the effect of aromatic:aliphatic ratio even in suboptimal molecular weight polypeptides. Firstly, it is noteworthy that, even in soluble regulation, there is a limit to the shortest time required to observe the effect on blood glucose in blood glucose droplets at approximately the same rate and fast time point. Secondly, each experimental group exhibited components of burst release, accompanied by the maximum changes observed in their constructs forming subcutaneous depots. This result suggests that during solubilization, a larger bolus volume reaches the bloodstream and is reduced when an equilibrium state is reached between depot release and protein clearance. Thirdly, each of our depot-forming formulations (GLP-1-[3Y:V]-20 and GLP-1-[Y:V]-20) controls blood glucose for at least an additional day compared to soluble RIDP controls.

[0165] By measuring the body weight of mice, we obtained supplementary information regarding the effectiveness of our subcutaneous depot (Figure 39). Again, we observed that our depot-forming protein exhibited a maximum burst release effect, resulting in the maximum change in body weight during the first two days. This effect appeared to be somewhat depot-dependent, with a lower limit of C sat The construct shows the greatest suppression of appetite. As expected, the saline injection has no effect on weight. Weight measurements also distinguish the two depot-forming fusions developed by the inventors from each other. High C sat The weight measurements of the structure show that their effectiveness declines by day 5, but low C sat The phenotypic effect appears to persist until the end of the study (day 8). These experiments mirrored similar results from optimal experiments using the GLP-1-ELP depot. While the recurrence of polypeptides with molecular weights exceeding 35 kDa decreased, an improvement was observed compared to glucose controls of 20–35 kDa. Therefore, we investigated the creation of larger molecular weight variants of the GLP-1-RIDP fusion.

[0166] The molecular weight of the polypeptide fusion was increased by successively reducing the aromatic content using aliphatic substitution, resulting in 0.5, 7, and 60 μM C sat This produced a series of fusions (Figure 40). Another GLP-1 protein fusion was also prepared using a 75% aliphatic content that does not exhibit UCST phase behavior under physiological conditions. The inventors also produced a C similar to GLP-1-[3Y:V]-40. sat A molecular weight control (GLP-1-[S]-20) was synthesized, which has a molecular weight of 7 μM compared to 12 μM, but half the molecular weight.

[0167] The blood glucose levels of mice with a 2.0 mg depot implanted in the subcutaneous space can be visualized in Figure 41. Various C sat These proteins exhibiting this characteristic also show varying releases from the depot in the subcutaneous space. The most hydrophobic depot, GLP-1-S-

[40] , appears to release only minimal amounts of the substance, suggesting that the biophysical properties of the depot delay the phenotypic effects of peptide drugs. 7–60 μM C sat GLP-1-[3Y:V]-40 and GLP-1-[Y:V]-40, which are median hydrophobic depots, exhibit similar levels of glucose control, representing a nearly complete 24-hour improvement over the smaller molecular weight version. The most hydrophilic “depot,” predicted to be soluble, is only able to control the glucose control over the first 24 hours. This is still an improvement over soluble controls with smaller molecular weights. These experiments support previous conclusions regarding the optimal depot design, which shows that the optimal release kinetics are achieved with 7–60 μM C sat We have identified that this can be achieved using polypeptides that exhibit this property. C for blood glucose sat The molecular weight-dependent effect can be visualized in Figure 42. Although the molecular weight increases, C remains within the optimal release range. sat Maintaining this state allows for an extension of glucose control for up to two more days. This effect is likely a result of delayed dispersion into the bloodstream, which extended the drug half-life due to delayed renal clearance.

[0168] Tracking of mouse body weight supports the conclusions inferred from blood glucose measurements (Figure 43). Here, the parabolic effect of depot hydrophobicity is evident at the hydrophobic and hydrophilic poles, with less burst release and shorter duration of mass regulation. The "optimal" constructs, GLP-1-[3Y:V]-40 and GLP-1-[Y:V]-40, still show mass regulation at day 7, suggesting that there must be a small amount of material being released from the depot even 144 hours after transplantation. The molecular weight control, GLP-1-[S]-20, shows less burst release and shorter duration of efficacy than its larger molecular weight analog, GLP-1-[3Y:V]-40, again supporting the conclusion of delayed entry into the bloodstream and prolonged duration from larger molecular weight depots.

[0169] In summary, the inventors designed a mimic fusion using the GLP-1-ELP system. They also identified several novel pathways for establishing a subcutaneous depot with an undesirable transition temperature. Using these two inventions, the inventors were able to create a depot that regulates blood glucose in vivo for up to 5 days in a DIO mouse model and acts with efficacy similar to conventional GLP-1-ELP depots.

Claims

1. (X-Z 1 -X-Z 2 -Z 3 -X-Z 4 -Z 3 ) n (In the formula, X is either proline (P) or glycine (G), and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z 2 Here, Asp(D), Arg(R), and Glu(E) are the vectors, where the ratio R:D can be any number, and D:E can be any number. Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio among N:Q:S:T can be any number, Z 4 (These are tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number.) A polypeptide having controlled reversible phase separation, comprising 10 or more repeat sequences of an amino acid sequence containing [specific amino acid sequence].

2. The polypeptide according to claim 1, wherein X is proline (P) or glycine (G), and the ratio of P:G is 1:3 to 3:

1.

3. Z 1 The polypeptide according to claim 1, wherein is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D not exceeding 1:5, and the ratio of K:R can be any number.

4. The polypeptide according to claim 1, wherein the phase separation depends on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration.

5. The polypeptide according to claim 1, wherein n is 10 to 200.

6. The polypeptide according to claim 1, wherein the molecular weight is at least 5 kDa to 500 kDa.

7. The polypeptide according to claim 1, wherein the molecular weight is approximately 5 kDa to approximately 100 kDa.

8. The polypeptide according to claim 1, wherein the phase separation temperature is 0 to 100°C.

9. The polypeptide according to claim 1, wherein the phase separation temperature is 4 to 25°C, about 25°C, 25 to 37°C, about 37°C, 35 to 38°C, or >38°C.

10. The polypeptide according to claim 1, comprising a modified amino acid, a reporter protein, or an enzyme.

11. The array is, (G-R-G-D-S-P-Y-S) m The polypeptide according to claim 10, comprising (wherein m is 20 to 80).

12. Sequence numbers 1-1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 The polypeptide according to claim 1, comprising an array selected from 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197 to 279, or one or more combinations thereof.

13. (X-Z 1 -X-Z 2 -Z 3 -X-Z 4 -Z 3 ) n (In the formula, X is either proline (P) or glycine (G), and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z 2 Here, Asp(D), Arg(R), and Glu(E) are the vectors, where the ratio R:D can be any number, and D:E can be any number. Z 3 These are asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number. Z 4 A pharmaceutically acceptable composition comprising a polypeptide having controlled reversible phase separation comprising a repeat sequence of 10 or more amino acid sequences (where Y:H:W:F:M:V:I:A:L is any number).

14. The composition according to claim 13, wherein X is proline (P) or glycine (G), and the ratio of P:G is 1:3 to 3:

1.

15. Z 1 The composition according to claim 13, wherein is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D not exceeding 1:5, and the ratio of K:R being any number.

16. Antimicrobial peptides selected from the following: antibody-binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelicidine-1 (SEQ ID NO: 169), A (A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (sequence number The composition according to claim 13, further comprising a binding molecule containing one or more of the following: (Sequence No. 177); fluorescent reporters (sfGFP (Sequence No. 179), mRuby3 (Sequence No. 181); RNA-binding proteins (PUM-HD (Sequence No. 183), eIF4E (Sequence No. 185), PABP (Sequence No. 187), Tis11D (Sequence No. 189)); KH domains (Yifan or FMRP (Sequence No. 191)); or AAV-binding peptides PKD1 (Sequence No. 193) or PKD2 (Sequence No. 195).

17. The composition according to claim 13, which enhances the bioavailability of the bound molecules compared to the bound molecules in their free form.

18. The composition according to claim 13, which enhances the recombination expression of the bound molecule compared to the bound molecule in its free form.

19. The composition according to claim 13, which enhances the stability of the binding molecule compared to the free form of the binding molecule.

20. The composition according to claim 19, which enhances the stability of the binding molecule during expression in a prokaryote or eukaryote compared to the free form of the binding molecule.

21. The composition according to claim 19, wherein the enhanced stability includes resistance to denaturation during freezing, thawing, freeze-drying, or long-term storage at temperatures above 4°C.

22. The composition according to claim 13, which modulates enzyme function, metabolic function, or physiological function within a cell or organism.

23. The composition according to claim 22, wherein the adjustment reduces the bioavailability of the binding molecule.

24. The composition according to claim 23, wherein the binding molecule comprises a therapeutic or cytotoxic protein or peptide.

25. A method for enhancing the bioavailability or stability of a protein, One or more proteins, (X-Z 1 -X-Z 2 -Z 3 -X-Z 4 -Z 3 ) n (In the formula, X is either proline (P) or glycine (G), and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K), where the ratio of R:D is any number, and the ratio of K:R can be any number; Z 2 Here, Asp(D), Arg(R), and Glu(E) are the vectors, where the ratio R:D can be any number, and D:E can be any number. Z 3 These are asparagine (N), glutamine (Q), serine (S), or threonine (T), and the ratio between N:Q:S:T can be any number. Z 4 (These are tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L), and the ratio between Y:H:W:F:M:V:I:A:L can be any number.) A method comprising producing a polypeptide fusion protein having controlled reversible phase separation, comprising 10 or more repeat sequences of an amino acid sequence containing [a specific amino acid sequence].

26. The method according to claim 25, wherein X is proline (P) or glycine (G), and the ratio of P:G is 1:3 to 3:

1.

27. Z 1 The method according to claim 25, wherein is arginine (R), aspartic acid (D), or lysine (K), the ratio of R:D not exceeding 1:5, and the ratio of K:R being any number.

28. The protein is an antimicrobial peptide selected from the antibody-binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelicidine-1 (SEQ ID NO: 169), A (A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, The method according to claim 25, comprising one or more of the following: GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA-binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domain (Yifan or FMRP (SEQ ID NO: 191)); or AAV-binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195).

29. The method according to claim 25, wherein the enhanced bioavailability of the fusion protein can be used for the isolation or separation of biological molecules.

30. The method according to claim 25, wherein the biological molecule comprises one or more lipids, cells, proteins, nucleic acids, carbohydrates, or virus particles.

31. The method according to claim 30, wherein the nucleic acid is single-stranded or double-stranded DNA or RNA.

32. The method according to claim 30, wherein the virus particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measles virus particle, or a herpesvirus particle.

33. The method according to claim 30, wherein the protein comprises albumin, a monoclonal IgG antibody, or an Fc fusion protein.

34. The method according to claim 30, wherein isolation or separation is achieved via reversible phase separation.

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