Charge-reversed N-terminal spider silk protein domains and uses thereof

Abstract

A protein comprising a portion of 100 to 160 amino acid residues having at least 70% identity to the N-terminal (NT) fragment of a spider silk protein, wherein the amino acid residue corresponding to position 40 of the NT is selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 of the NT is selected from the group consisting of Asp and Glu, said protein being useful as part of a fusion protein for improving the solubility of a desired protein or polypeptide that is another part of the fusion protein.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to the field of proteins and polypeptides, more specifically to the expression and production of spider silk proteins (spidroins) and other non-spidroin proteins and polypeptides. The present invention provides novel proteins, useful by themselves and as part of novel fusion proteins for the expression and production of desired proteins and polypeptides, as well as nucleic acid molecules encoding these novel proteins and fusion proteins. The present invention also provides methods for the expression and production of desired proteins or polypeptides. [Background technology]

[0002] 2. Background of the Invention Although the production of proteins and polypeptides from DNA can be achieved in a variety of hosts, a common problem is the formation of insoluble protein / polypeptide aggregates, which can seriously hinder or even prevent the production of functional proteins / polypeptides. This problem is typically exacerbated with low solubility proteins and polypeptides, such as membrane-associated proteins and polypeptides.

[0003] Membrane-associated proteins represent 20-30% of the cellular proteome and are the target of many currently available drugs. To be inserted into a membrane, a protein requires at least one stretch of 15-20 amino acid residues that promotes membrane insertion according to the biological hydrophobicity scale. At the same time, the hydrophobicity of amino acid side chains is a key determinant of aggregation potential, and hydrophobic amino acid residues (Val, Ile, Phe and Cys) promote β-sheet formation and are overrepresented in amyloids that form the core region of many disease-related proteins. Thus, membrane-associated proteins are prone to aggregation, which can seriously impede or even prevent the production of functional recombinant proteins.

[0004] For example, pulmonary surfactant protein C (SP-C) is a transmembrane (TM) protein that is difficult to recombinantly produce due to its extremely hydrophobic nature. SP-C is produced by alveolar type II cells and is a component of the surface active agent necessary to prevent alveolar collapse at the end of expiration. Newborns often suffer from respiratory distress due to insufficient amounts of surfactant. Today, this condition is treated with surfactant preparations extracted from animal lungs, such as Curosurf®, Infasurf®, Alveofact®, and Survanta®. Treatment with exogenous surfactant is also potentially beneficial for adult patients with respiratory distress, but the supply of surfactant is very limited and very expensive. Surfactant preparations based on peptides produced in heterologous systems would be superior to the natural extracts (and chemically synthesized peptide-containing preparations) used today due to lower production costs and higher production yields. It would also be advantageous from a regulatory perspective.

[0005] SP-C33Leu is a mutant of SP-C in which the N-terminal portion is truncated by two residues, two Cys residues are replaced by Ser, one Leu residue is replaced by Lys, one Met residue is replaced by Leu, and the membrane-spanning residues (usually mainly Val) are replaced by Leu to increase the stability of the transmembrane helix. KL4 is another surfactant analogue designed to mimic the properties of pulmonary surfactant protein B (SP-B) and consists of repeated repeats of Lys-Leu-Leu-Leu-Leu. SP-C33Leu and KL4 reproduce the function of natural surfactant peptides containing transmembrane inserts but are less prone to aggregation and therefore may be possible to produce in large quantities for the development of synthetic surfactant formulations. Both peptides can be produced by chemical synthesis, but the process generates by-products that may be difficult to remove and characterize.

[0006] Pulmonary surfactant proteins A (SP-A) and D (SP-D) do not insert into the membrane, but rather play a role in the pulmonary immune response via their carbohydrate-binding domain. They are large soluble protein complexes involved in the first line of defense of the lungs and regulate the function of innate immune cells (e.g. macrophages) as well as adaptive immune cells. The proteins belong to the collectin family of C-type lectins consisting of an N-terminal collagen-like region and a C-terminal calcium-dependent carbohydrate recognition domain. In their functional form, the proteins are arranged as trimeric polypeptide chains via their N-terminal region and further assembled into larger oligomers of different shapes. SP-A consists of six trimeric subunits arranged like a "bouquet", while SP-D arranges as a cross of four trimeric subunits. Although the proteins are hydrophilic, they are averse to recombinant production and have been expressed as insoluble inclusion bodies and purified by denaturation and refolding. Currently, surfactant preparations in clinical use do not contain SP-A or SP-D, and it is of interest to investigate whether the addition of these components, which are naturally part of the surfactant, would improve current surfactant therapy. Human SP-A and SP-D can be isolated from patients with pulmonary alveolar proteinosis or from amniotic fluid, but the yields are low and the oligomeric state is heterogeneous. Recombinant production of the proteins would allow for scale-up and reproducible manufacture for therapeutic use, but attempts to date have been unconvincing.

[0007] Other examples of proteins and polypeptides that cause problems when expressed from recombinant DNA are Aβ-peptide, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF and actin; SP-B, α-defensin and β-defensin; class AH apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; avidin and streptavidin; protease 3C; and immunoglobulins and fragments thereof.

[0008] One solution to this problem is to express the desired protein or polypeptide as a fusion protein with a solubility enhancing peptide / domain, i.e., the protein or polypeptide that provides the required solubility. The fusion protein may be cleaved and the desired protein isolated. Alternatively, the desired protein / polypeptide may be kept integrated into a soluble fusion protein, where it remains functional and can be subjected to further characterization, e.g., activity and interaction studies, structure determination and crystallization. Thioredoxin (Trx) is one of the most widely used solubility enhancing fusion partners that accumulates to high levels in the E. coli cytoplasm and has been shown to dramatically increase the solubility of many heterologous proteins and small peptides. Another successful fusion partner is the immunoglobulin binding domain B1 from streptococcal protein G (PGB1). The high stability and small size (56 residues) of this domain provide excellent qualities for the expression of small domains and peptides, as well as for downstream structural characterization.

[0009] WO 2011 / 115538 discloses fusion proteins comprising a solubility enhancing moiety derived from the N-terminal (NT) fragment of a spider silk protein and a moiety which is a desired protein or polypeptide. A pH above 6.4 is preferred to prevent aggregation of the solubility enhancing moiety.

[0010] EP 2 644 619 also discloses a fusion protein comprising a solubility enhancing moiety derived from the N-terminal (NT) fragment of a spider silk protein and a moiety which is a desired protein or polypeptide, the solubility enhancing moiety being constitutively monomeric below pH 6.4 but not increasing the expression level of the resulting fusion protein compared to the wild-type NT fragment.

[0011] Despite these advances in the field, fusion protein approaches have limitations with respect to product expression, stability and solubility. The use of fusion partners in large-scale heterologous protein production is not common, primarily due to the need for additional expensive chromatography steps and / or the difficulty of removing the fusion partner. Summary of the Invention

[0012] Summary of the Invention It is an object of the present invention to provide a fusion partner, i.e. a portion of a fusion protein, which is useful for increasing the solubility of a desired protein or polypeptide, which is another portion of said fusion protein.

[0013] It is also an object to provide methods for novel proteins and improved recombinant production methods for hydrophobic proteins and polypeptides and proteins and polypeptides having hydrophobic regions.

[0014] It is a further object of the present invention to provide fusion partners that are stable monomers over a wide pH range.

[0015] Another object of the present invention is to provide a convenient method for producing and isolating a desired protein or polypeptide, including expression in a fusion protein.

[0016] For these and other objects which will become apparent from the following specification, the present invention provides, according to a first aspect, a protein as claimed in the accompanying claims. The invention is generally based on the discovery that this charge-reversed NT mutant is unable to dimerize, is stabilized, hypersoluble and allows efficient production of membrane-spanning and aggregation-prone proteins. The fusion protein according to the invention can advantageously form micelles.

[0017] According to a second aspect, the present invention provides the use of this protein as part of a fusion protein to improve the solubility of a desired protein or polypeptide which is another part of the fusion protein.

[0018] According to a third aspect, the present invention provides a method for the production of a desired protein or polypeptide as claimed in the accompanying claims.

[0019] [Table 1-1] [Table 1-2] [Table 1-3] [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a sequence alignment of spidroin N-terminal domains. [Diagram 2] FIG. 2 shows the ratio of fluorescence emission at 339 nm and 351 nm versus pH for NTD40K / K65D and wild-type NT. [Diagram 3] FIG. 3 is a comparison of NTwt and NTD40K / K65D using 2D HSQC NMR. [Figure 4]FIG. 4 shows urea-induced unfolding of NTwt and NTD40K / K65D as a measure of stability. [Diagram 5] FIG. 5 shows the thermal stability of NTwt and NTD40K / K65D as measured by CD spectroscopy. [Figure 6] FIG. 6 shows the refolding ability of NTwt and NTD40K / K65D measured by CD spectroscopy. [Figure 7] Figures 7-8 are SDS-PAGE evaluations of the fusion proteins and the resulting purified proteins and peptides. [Figure 8] Figures 7-8 are SDS-PAGE evaluations of the fusion proteins and the resulting purified proteins and peptides. [Figure 9] FIG. 9 shows the ESI-MS spectrum of SP-C33Leu produced by the method of the present invention. [Figure 10] FIG. 10 shows the effect of rSP-C33Leu on tidal volume. [Figure 11] FIG. 11 shows the effect of rSP-C33Leu on lung volume. [Figure 12] FIG. 12 is an SDS-PAGE evaluation of the β17 fusion protein and the resulting purified polypeptide. [Figure 13] FIG. 13 shows gel filtration of soluble NTD40K / K65D-β17 fusion protein. [Figure 14] FIG. 14 is a graph of ThT fluorescence showing fibrillization of β17 following proteolysis of the fusion protein with thrombin. [Figure 15] FIG. 15 is an SDS-PAGE assessment of Bri2 BRICHOS fusion protein expression and solubility. [Figure 16] FIG. 16 is an SDS-PAGE analysis of Bri2 BRICHOS purification. [Figure 17] FIG. 17 shows gel filtration to determine the oligomeric state of the Bri2113-231 fusion protein. [Figure 18]FIG. 18 shows SDS-PAGE analysis of hCAP18 expressed in fusion with NTwt or NTD40K / K65D. [Figure 19] FIG. 19 shows a SEC chromatogram of the NTD40K / K65D-SP-C33Leu fusion protein. [Figure 20] FIG. 20 shows a TEM micrograph of negatively stained NTD40K / K65D-containing proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The present invention relates to the production and expression of proteins and polypeptides. Depending on the purpose of this production, the final product may be different. For example, it may be desirable to obtain the desired protein or polypeptide inserted into a lipid membrane, in solution or in association with other biomolecules. It shall be recognized that it may also be highly desirable to obtain the desired protein or polypeptide as part of a fusion protein, which may provide a suitable handle for purification and detection and / or provide desired properties, such as stability and solubility. Maintaining the desired protein or polypeptide functionally integrated in a soluble fusion protein is useful for characterizing and studying the desired protein or polypeptide.

[0022] The present invention is generally based on the insight into the utility of certain variants of the N-terminal (NT) fragment of spider silk proteins due to their surprising ability to exist as soluble monomers regardless of the pH of the surrounding aqueous medium, as well as their superior properties as fusion partners due to their exceptionally high solubility.

[0023] Spider silk consists primarily of large aggregation-prone proteins (spidroins) produced in the spider's abdominal gland. They are constructed from extensive and predominantly hydrophobic stretches of repeating alanine- and glycine-rich segments flanked by globular and hydrophilic domains at their N-terminal (NT) and C-terminal (CT) ends. During spinning, spidroins pass through narrow ducts to convert from soluble proteins to solid fibers in a process that involves precise control of environmental conditions, including level of hydration, shear forces, ionic composition, pH and carbon dioxide pressure. Despite their aggregation-prone nature, spidroins are stored in remarkably high concentrations (30-50% w / w) within the spider silk gland.

[0024] The NT domain is the most conserved part of spidroins and folds into a soluble ~130 residue five-helical bundle with a dipolar charge distribution. Furthermore, the NT has a crucial role in fiber formation, as it forms antiparallel dimers at pH below 6.5, which is thought to be a key step for interconnecting spidroin micelles within the spinning duct.

[0025] In the known spider MaSp1 and MaSp2 silk protein species (see, for example, FIG. 1), D40 and K65 of the NT portion (SEQ ID NO: 9) are conserved and mediate intersubunit electrostatic interactions by salt bridges between the negatively charged D40 and positively charged K65 side chains, which stabilize the dimer structure. Similarly, there are corresponding oppositely charged amino acid residues in the known minor ampullate spider silk (MiSp) species. It has now been realized and demonstrated in the examples that charge reversal mutations of these residues to provide a positively charged (basic) amino acid residue at position 40 and a negatively charged (acidic) amino acid residue at position 65 surprisingly eliminate the intersubunit salt bridge between these positions, which in turn has a significant effect on the pH-dependent dimerization ability of the NT fragment. As a result of these charge-reversal mutations, the NT mutants surprisingly become stable monomers over a wide pH interval and achieve superior properties as fusion partners with desired proteins or polypeptides, including high expression, high stability and pH insensitivity of the resulting fusion proteins. The NT mutants are highly soluble in water. The high expression levels of fusion proteins obtained with the charge-reversal NT mutants of the present invention are comparable to those of another known NT mutant, NT A72R NT of the corresponding fusion protein wt This is particularly surprising considering that the expression levels achieved by the charge-reversal mutant NTs according to the present invention are lower than that of the control. Without wishing to be bound by any particular scientific theory, it is suggested that the improved stability and refolding ability of the charge-reversal mutant NTs according to the present invention can be explained by a lesser dipole charge distribution, which reduces unstable charge clusters.

[0026] The yield of soluble fusion protein after expression in E. coli is typically at least 2-fold higher compared to conventional tag-like thioredoxin and PGB1, and the fusion protein can be purified to homogeneity, for example by salt precipitation and / or precipitation in organic solvents. The cleaved desired protein or peptide is active, for example in animal models of disease. The charge-reversed NT mutants are unable to dimerize, are stabilized and ultra-soluble, allowing higher fusion protein yields as well as the production of aggregation-prone proteins for which recombinant production was previously ineffective.

[0027] Without wishing to be limited to a particular theory, the experimental results disclosed herein support that recombinant production of a desired peptide or protein can be enhanced using the charge-reversed NT mutants of the present invention as fusion tags that mediate solubility and shield hydrophobic regions of the desired protein from the surrounding aqueous solution within micelle-like particles. D40K / K65D NT is unable to dimerize at low pH due to its reduced dipole charge distribution and is therefore able to mediate solubility over a wider pH range. D40K / K65D Size-exclusion chromatography of -SP-C33Leu (Figure 19) shows that the purified amphiphilic fusion protein arranges into assemblies of 510 kDa, and micelle-like particles of approximately 10-15 nm in size are observed by TEM (Figure 20).

[0028] Thus, the formation of micelles or micelle-like particles comprising or consisting of fusion proteins comprising the charge-reversed NT mutants of the invention is believed to be a useful intermediate structure for the recombinant production of desired proteins and polypeptides, particularly hydrophobic proteins and polypeptides and proteins and polypeptides having hydrophobic regions, such as membrane proteins and polypeptides and membrane-associated proteins and polypeptides. Micelles typically have a size in the range of 5-100 nm, e.g. 5-30 nm, and preferably 5-20 or 10-15 nm.

[0029] Membrane-associated proteins and many other commercially relevant proteins are difficult to produce and purify in their native form due to their hydrophobic and aggregation-prone properties. The lack of a sustainable and general production system makes it difficult to manufacture and evaluate these proteins, for example as pharmaceuticals. The charge-reversed NT mutants according to the invention, which do not have the ability to dimerize, demonstrated improved stability and refolding ability and were used as highly soluble fusion partners for the production of surfactant peptide analogues SP-C33Leu, KL4, surfactant proteins SP-A and SP-D and their truncated variants, amyloidogenic polypeptides β17, Aβ and IAPP, hCAP18, Nicastrin, eGFP and Bri2-BRICHOS protein domains. The amount of fusion protein obtained was up to 8-fold higher compared to the PGB1 fusion protein, and all peptides / proteins could be produced as soluble and functional proteins after removal of the NT domain.

[0030] The use of fusion partners in large-scale heterologous protein production is uncommon, mainly due to the need for additional expensive chromatography steps and / or the difficulty of removing the fusion partner. However, NT and charge-reversed NT allow efficient purification of hydrophobic target peptides with a simple NaCl precipitation and ethanol extraction step that avoids the need for chromatography. The procedure described herein represents an inexpensive, efficient and, from a regulatory point of view, advantageous method for the production of proteins, such as non-animal derived SP-C33Leu for clinical use in synthetic pulmonary surfactants. It is shown herein that recombinant SP-C33Leu is identical to the synthetic peptide, and that a mixture of recombinant SP-C33Leu and synthetic phospholipids is identical to the porcine derived surfactant Curosurf®, in terms of function in animal models of respiratory distress.

[0031] The surfactant proteins SP-A and SP-D do not insert into the membrane, but rather play a role in the pulmonary immune response via their carbohydrate-binding domains. Although the proteins are hydrophilic, they are reluctant to be produced recombinantly. The use of charge-reversed NTs as fusion partners allows high expression and subsequent purification of soluble derivatives of SP-A and SP-D. This indicates that charge-reversed NTs can serve as general solubility-enhancing fusion partners for biotechnological applications, enabling the heterologous production of TM peptides as well as proteins that are difficult to produce today due to their hydrophobicity or tendency to aggregate.

[0032] Thus, the present invention provides, according to a first aspect, a protein comprising a portion of 100-160 amino acid residues having at least 70% identity with SEQ ID NO: 1 or SEQ ID NO: 96, wherein the amino acid residue corresponding to position 40 of SEQ ID NO: 1 is a basic amino acid residue selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 of SEQ ID NO: 1 is an acidic amino acid residue selected from the group consisting of Asp and Glu. Although wild-type NT is highly water-soluble and useful as a solubility-enhancing moiety in, for example, fusion proteins for expression of desired proteins or polypeptides, it also forms dimers in the pH interval of 5.5-7.2, which increases the risk of undesired aggregation of the fusion protein. This is a pH interval useful for the functionality and stability of certain desired proteins and polypeptides. It is also a pH interval useful for certain purification protocols, for example when ion exchange, such as cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) is used as the purification principle. It is also a pH interval useful for certain expression hosts, such as yeast. It has now been realized that the charge-reversed double mutant NT of the present invention reduces the ability of a protein to form dimers without adversely affecting the solubility or ability of a desired protein or polypeptide to increase its solubility when linked in a fusion protein. The mutant NT protein of the present invention is therefore useful as such for studying the physiologically relevant NT monomer itself. The mutant NT protein of the present invention is also useful as a solubility-enhancing moiety in fusion proteins, since it reduces the risk of undesired aggregation of the fusion protein, thereby opening a new pH window (5.5-7.2), in which the charge-reversed double mutant NT from spider silk proteins can be used in biochemical applications where protein / polypeptide monomer solubility in aqueous solution is desired, e.g. for the production or characterization of a desired protein or polypeptide.Surprisingly, experimental data has determined that these changes at positions 40 and 65 do not alter the net charge of the resulting protein, but do improve its ability to provide stability and solubility to the desired protein / polypeptide moiety to which it is fused.

[0033] In a preferred embodiment, the fusion protein according to the invention is present as a micelle or micelle-like particle. Micelles typically have a size in the range of 5-100 nm, e.g. 5-30 nm, 5-20 nm, or 10-15 nm. This intermediate micellar structure is believed to support high stability and solubility, and ultimately high yield, of fusion proteins, particularly of hydrophobic proteins and polypeptides and fusion proteins comprising proteins and polypeptides with hydrophobic regions, and thus protect water-insoluble peptides during expression and purification in aqueous solvents. Micelles typically have a size in the range of 5-100 nm, e.g. 5-30 nm, and preferably 5-20 nm or 10-15 nm.

[0034] In a preferred embodiment, the amino acid residue corresponding to position 40 of SEQ ID NO: 1 is Lys or Arg, and preferably Lys. In a preferred embodiment, the amino acid residue corresponding to position 65 of SEQ ID NO: 1 is Asp. Six possible preferred variants for these two positions are presented as SEQ ID NOs: 2 to 7. A particularly preferred variant is SEQ ID NO: 2, in which the amino acid residue corresponding to position 40 of SEQ ID NO: 1 is Lys; and the amino acid residue corresponding to position 65 of SEQ ID NO: 1 is Asp. For the avoidance of doubt, the corresponding mutation positions in SEQ ID NOs: 2 to 7 are positions 36 and 61, due to the shorter N-terminus.

[0035] In a preferred embodiment, the amino acid residue corresponding to position 72 of SEQ ID NO:1 is not Arg. It is preferably an uncharged residue, i.e. it is not Lys, Arg, His, Glu or Asp. In a particular preferred embodiment, the amino acid residue corresponding to position 72 of SEQ ID NO:1 is selected from the group consisting of Ala, Val, Phe, Pro, Leu, Ile, Trp, Met, Cys and Gly; and preferably Ala, Val, Leu, Ile and Gly. In a preferred embodiment, the amino acid residue corresponding to position 72 of SEQ ID NO:1 is Ala or Gly, preferably Ala.

[0036] As mentioned above, the moieties of the present invention are derived from spider silk proteins, or NT fragments of spidroins. Although the examples are necessarily directed to a protein derived from a particular NT fragment, in this case major spidroin 1 (MaSp1) from Euprosthenops australis, it is believed that the methods disclosed herein are applicable to any similar protein moiety. The terms "spidroins" and "spider silk proteins" are used interchangeably throughout the specification and encompass all known spider silk proteins, including: major ampullate spider silk proteins, typically abbreviated as "MaSp" or "ADF" in the case of Araneus diadematus, and minor ampullate spider silk proteins, typically abbreviated as "MiSp". Major ampullate spider silk proteins are generally of two types, 1 and 2. These terms further include the new NT protein fragments according to the invention as defined in the appended claims and in the individual embodiments, as well as other non-naturally occurring proteins having a high degree of identity and / or similarity to known spider silk NT protein fragments.

[0037] The moieties of the present invention have a high degree of similarity with the N-terminal (NT) amino acid sequences of spider silk proteins. As shown in FIG. 1, these amino acid sequences are well conserved among various species and spider silk proteins, including MaSp1, MaSp2 and MiSp. Therefore, a person skilled in the art is well aware of how and to what extent the amino acid sequence may be changed without departing from the properties and functions of the N-terminal spider silk protein fragment. Table 1 lists the spidroin NT fragments and other NT moieties of the present invention aligned in FIG. 1 as shown by the GenBank accession entries.

[0038] [Table 2]

[0039] Only the portion corresponding to the N-terminal domain is shown for each sequence, and the signal peptide is omitted. The Nc flag and Nlm flag were converted and edited according to Rising A. et al., Biomacromolecules 7, 3120-3124 (2006)).

[0040] It is not important which particular NT moiety is present in the protein of the invention, so long as the NT moiety is not completely absent. Thus, the NT moiety of the invention may be selected from the MaSp1 or MaSp2 amino acid sequence shown in FIG. 1, or a sequence having a high degree of similarity or a MiSp amino acid sequence, such as any one of SEQ ID NOs: 96-99. A variety of sequences may be used in the fusion protein of the invention. Based on the homologous sequences of FIG. 1, the following sequence constitutes the consensus MaSp NT amino acid sequence: QANTPWSSPNLADAFINSF(M / L)SA(A / I)SSSGAFSADQLDDMSTIG(D / N / Q)TLMSAMD(N / S / K)MGRSG(K / R)STKSKLQALNMAFASSMAEIAAAESGG(G / Q)SVGVKTNAISDALSSAFYQTTGSVNPQFV(N / S)EIRSLI(G / N)M(F / L)(A / S)QASANEV (SEQ ID NO: 10).

[0041] The sequence of the inventive moiety according to the present invention preferably has at least 50% identity, preferably at least 60% identity, with the consensus amino acid sequence SEQ ID NO: 10, which is based on the wild-type NT amino acid sequence of Figure 1. In a preferred embodiment, the sequence of the inventive moiety according to the present invention has at least 65% identity, preferably at least 70% identity, with the consensus amino acid sequence SEQ ID NO: 10. In a preferred embodiment, the solubility-enhancing moiety according to the present invention further has 70%, preferably 80% similarity to the consensus amino acid sequence SEQ ID NO: 10.

[0042] A representative inventive moiety according to the present invention is SEQ ID NO:2 (encoded by SEQ ID NO:8), which is derived from the Euprosthenops australis NT moiety (SEQ ID NO:9) with a substitution of aspartic acid at position 40 with lysine or any other basic residue, and a substitution of lysine at position 65 with aspartic acid or any other acidic residue, as described hereinabove and in SEQ ID NO:1. According to a preferred embodiment of the present invention, the inventive moiety further has at least 70% identity, such as at least 75% identity, preferably at least 80% identity, with SEQ ID NO:1, or any of SEQ ID NOs:96-99, or any individual amino acid sequence in FIG. 1. In a preferred embodiment of the present invention, the inventive moiety has at least 85%, such as at least 90% or even 95% identity, with SEQ ID NO:1, or any of SEQ ID NOs:96-99, or any individual amino acid sequence in FIG. 1. In a preferred embodiment of the invention, the solubility-enhancing moiety is identical to SEQ ID NO:9, or any of SEQ ID NOs:96-99, or any individual amino acid sequence in Figure 1, provided that the acidic residue at position 40 and the basic residue at position 65 (or possible corresponding positions) are substituted as described herein above.

[0043] The term "% identity" as used throughout the specification and appended claims is calculated as follows: A query sequence is aligned to a target sequence using the CLUSTAL W algorithm (Thompson, JD, Higgins, DG and Gibson, TJ, Nucleic Acids Research, 22: 4673-4680 (1994)). Comparisons are made over the shortest corresponding window of the aligned sequences. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as the % identity.

[0044] The term "% similarity" as used throughout the specification and appended claims is calculated as described for "% identity" below, with the exception that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys are similar; the basic residues Lys, Arg and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic uncharged residues Gln, Asn, Ser, Thr and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

[0045] Throughout this description, alternative embodiments of the present invention will meet the corresponding percentage of similarity instead of the specified percentage of identity. Other alternative embodiments will meet the specified percentage of identity, as well as another higher percentage of similarity selected from the group of preferred percentages of identity for each sequence. For example, a sequence may be 70% similar to another sequence; or a sequence may be 70% identical to another sequence; or a sequence may be 70% identical and 90% similar to another sequence.

[0046] Moieties of the invention comprise between 100 and 160 amino acid residues. Moieties of the invention preferably comprise at least 100, or more than 110, preferably more than 120 amino acid residues. Moieties of the invention also desirably comprise a maximum of 160, or less than 140 amino acid residues. A typical moiety of the invention comprises about 130 to 140 amino acid residues.

[0047] As detailed in WO 2011 / 115538 (incorporated herein in its entirety), N-terminal (NT) fragments of spider silk proteins are particularly useful as solubility-enhancing moieties in fusion proteins produced from recombinant DNA. According to a further aspect, the invention is further based on the insight of the usefulness of the charge-reversed double mutant NT according to the invention in such fusion proteins due to its ability to exist as a soluble monomer regardless of the pH of the surrounding aqueous medium. The resulting fusion proteins are surprisingly stable and can be produced in high yields.

[0048] According to this aspect, the present invention provides a fusion protein comprising: (i) at least one solubility-enhancing moiety of 100-160 amino acid residues having at least 70% identity to SEQ ID NO:1 or SEQ ID NO:96, wherein the amino acid residue corresponding to position 40 of SEQ ID NO:1 is selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 of SEQ ID NO:1 is selected from the group consisting of Asp and Glu; and (ii) at least one moiety which is a desired protein or polypeptide. Preferred features of the solubility-enhancing moiety are set forth herein above. Surprisingly, it has been determined from experimental data that these changes at positions 40 and 65 of the solubility-enhancing moiety do not alter the net charge of the resulting fusion protein, but improve its ability to provide stability and solubility to the desired protein / polypeptide moiety to which it is fused, and surprisingly, dimerization is prevented by the solubility-enhancing moiety of the fusion protein, since electrostatic interactions involving oppositely charged residues 40 and 65 are not formed.

[0049] In a preferred embodiment, the fusion protein consists of: (i) at least one solubility enhancing moiety of 100-160 amino acid residues having at least 70% identity to SEQ ID NO: 1 or SEQ ID NO: 96, where the amino acid residue corresponding to position 40 of SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 of SEQ ID NO: 1 is selected from the group consisting of Asp and Glu; and (ii) at least one moiety which is a desired protein or polypeptide, optionally including other preferred features disclosed herein, such as a linker peptide and / or cleavage site between the solubility enhancing moiety and the desired protein or polypeptide. In experiments, high yields of different fusion proteins were achieved in E. coli. The fusion protein itself may be useful in isolated form, for example for the study of otherwise aggregated or poorly soluble proteins in soluble form, or in crystallization in conjunction with X-ray crystallography. The fusion protein may also be cleaved to release the desired protein or polypeptide.

[0050] The term "fusion protein" as used herein refers to a protein made by expression from a recombinant nucleic acid, i.e., DNA or RNA, which is artificially created by combining two or more nucleic acid sequences that do not normally occur together (genetic engineering). Fusion proteins according to the invention are recombinant proteins, therefore, they are not identical to naturally occurring proteins. The combined nucleic acid sequences code for different proteins, partial proteins or polypeptides with specific functional properties. The resulting fusion protein, or recombinant fusion protein, is a single protein that has functional properties derived from each of the original proteins, partial proteins or polypeptides.

[0051] In a particular embodiment, the fusion protein and the corresponding gene according to the invention are chimeric, i.e. the protein / gene fragment originates from at least two different species. The solubility enhancing moiety originates from an N-terminal fragment of a spider silk protein. According to this aspect, the desired protein or polypeptide is preferably a non-spidroin protein. This means that the desired protein or polypeptide is not derived from a C-terminal, repeat or N-terminal fragment of a spider silk protein. According to another aspect, the desired protein or polypeptide is preferably a spidroin protein. This means that the desired protein or polypeptide is derived from a C-terminal, repeat or N-terminal fragment of a spider silk protein. The desired protein or polypeptide may in turn also be a fusion between a spidroin protein moiety and a non-spidroin polypeptide or protein moiety. Typically, the spidroin protein moiety provides the ability to form ordered polymers, while the non-spidroin polypeptide or protein moiety may provide the desired affinity properties, e.g. cell-binding peptides, immunoglobulins and functional fragments thereof.

[0052] The fusion protein according to the invention may also comprise one or more linker peptides. The linker peptide(s) may be placed between the solubility enhancing moiety and the desired protein or polypeptide moiety, or may be placed at either end of the solubility enhancing moiety and the desired protein or polypeptide moiety. If the fusion protein comprises more than one solubility enhancing moiety, the linker peptide(s) may also be placed between the two solubility enhancing moieties. The linker(s) may provide a spacer between the functional units of the fusion protein, but may also constitute a handle for identification and purification of the fusion protein, e.g. His and / or Trx tags. If the fusion protein comprises more than one linker peptide for identification and purification of the fusion protein, they are preferably separated by a spacer sequence, e.g. His6-spacer-His6-. The linker may also constitute a signal peptide, e.g. a signal recognition particle substrate, which directs the fusion protein to the membrane and / or causes secretion of the fusion protein from the host cell into the surrounding medium. The fusion protein may also contain a cleavage site in its amino acid sequence, which allows for cleavage and removal of the linker(s) and / or the solubility enhancing moiety or moieties. Various cleavage sites are known to those skilled in the art, such as cleavage sites for chemical agents, such as CNBr after the Met residue, and hydroxylamine between the Asn-Gly residues, cleavage sites for proteases, such as thrombin or protease 3C, and self-splicing sequences, such as intein self-splicing sequences. A preferred cleavage site is after the Met residue.

[0053] Each solubility-enhancing moiety is linked directly or indirectly to the desired protein or polypeptide moiety. Direct linkage refers to a direct covalent bond between the two moieties without an intervening sequence, such as a linker. Indirect linkage also refers to the two moieties being linked by a covalent bond, but with the presence of an intervening sequence, such as a linker and / or one or more further solubility-enhancing moieties.

[0054] The at least one solubility enhancing moiety may be located at either end of the desired protein or polypeptide, i.e. at the C-terminus or at the N-terminus. It is preferred that the at least one solubility enhancing moiety is located at the N-terminus of the desired protein or polypeptide. If the fusion protein comprises one or more linker peptide(s) for identification and purification of the fusion protein, e.g. His or Trx tag(s), it is preferred that they are located at the N-terminus of the fusion protein. The at least one solubility enhancing moiety may also be integrated within the desired protein or polypeptide, e.g. between domains or parts of the desired protein. In a preferred embodiment, the at least one solubility enhancing moiety constitutes the N-terminus and / or C-terminus of the fusion protein, i.e. no linker peptide or other sequence is present at the end of the solubility enhancing moiety. A typical fusion protein according to the invention may comprise 1-6, such as 1-4, e.g. 1-2 solubility enhancing moieties.

[0055] In a preferred embodiment, the fusion protein comprises at least two solubility enhancing moieties, each derived from the N-terminal (NT) fragment of a spider silk protein as described herein above. The solubility enhancing moieties, preferably two solubility enhancing moieties, may be consecutively located at either end of the desired protein or polypeptide, i.e. located at the C-terminus or located at the N-terminus. The consecutively located solubility enhancing moieties may also be integrated within the desired protein or polypeptide, for example between domains or portions of the desired protein. The solubility enhancing moieties may also be non-consecutively located at either end of the desired protein or polypeptide, i.e. located at the C-terminus and the N-terminus, or may be located at one end of the desired protein or polypeptide and integrated within the desired protein or polypeptide. A typical preferred fusion protein according to the invention may comprise 2 to 6, such as 2 to 4, solubility enhancing moieties.

[0056] In a preferred embodiment, the fusion protein of the present invention has at least one cleavage site located between at least one desired protein or polypeptide moiety and at least one solubility enhancing moiety. This allows for cleavage of the fusion protein and purification of the desired protein. It should be noted, however, that it may be desirable to obtain the desired protein or polypeptide as part of the fusion protein, which may provide a suitable handle for purification and detection and / or provide desirable properties, such as stability and solubility. In this case, the cleavage site may be omitted, or alternatively, the cleavage site may be included but the cleavage step may be omitted.

[0057] A preferred fusion protein has the form of an N-terminally located solubility enhancing moiety coupled to a C-terminally located protein or polypeptide of interest by a linker peptide of 1-30 amino acid residues, e.g. 1-10 amino acid residues. The linker peptide may include a cleavage site. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may include a purification tag, e.g. a His tag, and a cleavage site.

[0058] Another preferred fusion protein has the form of an N-terminally located solubility enhancing moiety directly coupled to a C-terminally located protein or polypeptide of interest. Optionally, the fusion protein has an N- or C-terminal linker peptide, which may include a purification tag, e.g., a His tag, and a cleavage site.

[0059] One preferred fusion protein has the form of two consecutive N-terminally located solubility enhancing moieties coupled to a C-terminally located protein or polypeptide of interest by a linker peptide of 1-30 amino acid residues, e.g. 1-10 amino acid residues. The linker peptide may comprise a cleavage site. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may comprise a purification tag, e.g. a His tag, and a cleavage site.

[0060] Another preferred fusion protein has the form of two consecutive N-terminally located solubility enhancing moieties directly coupled to a C-terminally located protein or polypeptide of interest. Optionally, the fusion protein has an N- or C-terminal linker peptide, which may include a purification tag, e.g., a His tag, and a cleavage site.

[0061] In the context of the present invention, a desired polypeptide is understood to mean a polypeptide of 5 to 50 amino acid residues, preferably 10 to 50, 20 to 50 or 40 to 50 amino acid residues. Furthermore, in the context of the present invention, a desired protein is understood to mean a protein of more than 50 amino acid residues, for example more than 80 amino acid residues. Preferably, the desired protein comprises less than 500 amino acid residues, for example less than 300 or less than 200 amino acid residues. Preferred sizes of the desired polypeptide or protein are in the range of 4 to 50 kDa, for example 5 to 50 kDa, 4 to 45 kDa and 5 to 45 kDa, preferably 8 to 30 kDa, more preferably 4 to 20, 5 to 20 or 8 to 30 kDa.

[0062] In a preferred embodiment of the invention, the desired polypeptide or protein is hydrophobic and has an aliphatic index of 65 or more, preferably 70 or more, as determined by Protparam (http: / / web.expasy.org / protparam / ; Gasteiger E. et al.; Protein Identification and Analysis Tools on the ExPASy Server; (in) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607). Preferably, the percentage of hydrophobic residues in a polypeptide as defined by the Grand Average of Hydropathy (GRAVY) index (Kyte, J. and Doolittle, RF (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.) should be between -1 and 2, preferably between 1 and 2.

[0063] In a preferred embodiment, the desired protein or polypeptide is selected from the group consisting of amyloidogenic proteins and polypeptides, disulfide-containing proteins and polypeptides, apolipoproteins, membrane proteins and polypeptides, protein and polypeptide drugs and drug targets, aggregation-prone proteins and polypeptides, proteases, and immunoglobulins and fragments thereof. In a preferred embodiment, the desired protein or polypeptide is selected from the group consisting of amyloidogenic proteins and polypeptides, disulfide-containing proteins and polypeptides, apolipoproteins, membrane proteins and polypeptides, protein and polypeptide drugs and drug targets, aggregation-prone proteins and polypeptides, and proteases.

[0064] One preferred group of desired proteins or polypeptides are Aβ-peptides, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF, actin and β17; SP-B, mini-BLeu, α-defensins and β-defensins; class AH apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, SP-C33Leu, KL4, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones including EPO and GH, and growth factors including IGF-I and IGF-II; SP-A, SP-D and analogs thereof; Bri2-BRICHOS and Bri2 113 - 231 Its variants, including ; A preferred group of desired proteins or polypeptides consists of Aβ-peptides, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF, actin and β17; SP-B, mini-BLeu, α-defensins and β-defensins; class AH apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, SP-C33Leu, KL4, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones including EPO and GH, and growth factors including IGF-I and IGF-II; SP-A, SP-D and analogs thereof; Bri2-BRICHOS and Bri2 113 - 231 Its variants, including ; avidin and streptavidin; and protease 3C.

[0065] The amyloidogenic proteins and polypeptides of the present invention include proteins and polypeptides associated with disease and functional amyloid. Examples of amyloidogenic proteins and polypeptides include amyloid beta peptide (Aβ-peptide), islet amyloid polypeptide (amylin or IAPP), prion protein (PrP), α-synuclein, calcitonin, prolactin, cystatin, atrial natriuretic factor (ATF) and actin. A further example is the designed polypeptide β17. Examples of amyloidogenic proteins and polypeptides of the present invention are listed in Table 2.

[0066] [Table 3]

[0067] Examples of disulfide-containing proteins and polypeptides include surfactant protein B (SP-B) and its variants, such as Mini-B, Mini-B27, Mini-BLeu, α-defensin, and β-defensin. Without being limited to a particular theory, it is contemplated that the solubility-enhancing moieties promote the formation of the desired intrachain disulfide bonds over interchain disulfide bonds in defensins and other disulfide-containing proteins and polypeptides.

[0068] Examples of disulfide-containing proteins and polypeptides according to the present invention are listed in Table 3.

[0069] [Table 4]

[0070] Examples of apolipoproteins include class AH apolipoproteins. Examples of apolipoproteins according to the present invention are listed in Table 4.

[0071] [Table 5]

[0072] Examples of membrane proteins and polypeptides include membrane-associated receptors, including cytokine receptors, KL4, LL-37, hCAP18, surfactant protein C (SP-C) and variants thereof, such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu. Other specific examples include Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL or SP-B proteins, optionally with a linker, such as Gly. n , Leu n , Gly-Ala n and the like. SP-C33Leu can be located N-terminally or, preferably, C-terminally to the Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL or SP-B protein.

[0073] Examples of membrane proteins and polypeptides according to the invention are listed in Table 5.

[0074] [Table 6]

[0075] Examples of protein and polypeptide drugs and drug targets include recombinantly produced hormones, including peptide and protein hormones such as erythropoietin (EPO) and growth hormone (GH), cytokines, growth factors such as insulin-like growth factors (IGF-I and IGF-II), KL4, LL-37, hCAP18, surfactant protein C (SP-C) and its variants such as SP-C(Leu), SP-C33, SP-C30, SP-C33Leu. Other specific examples include Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL or SP-B proteins, optionally with a linker, such as Gly. n , Leun , Gly-Ala n and SP-C33Leu fused via a nucleotide sequence such as nucleotide sequence 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL, or SP-B protein. SP-C33Leu may be located N-terminally or, preferably, C-terminally to the Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL, or SP-B protein. Further preferred examples include surfactant protein A (SP-A) and D (SP-D), and analogs thereof. Further preferred examples include Bri2-BRICHOS and Bri2 113 - 231 and variants thereof, including nicastrin.

[0076] Examples of protein and polypeptide drugs and drug targets according to the present invention are listed in Table 6.

[0077] [Table 7-1] [Table 7-2]

[0078] Examples of proteins and polypeptides prone to aggregation include avidin, streptavidin and the extracellular ligand binding sites of cytokine receptors.

[0079] Examples of aggregation-prone proteins and polypeptides according to the present invention are listed in Table 7.

[0080] [Table 8]

[0081] Examples of proteases include protease 3C from Coxsackievirus or human rhinovirus. Further examples of proteases according to the present invention are listed in Table 8.

[0082] [Table 9]

[0083] In a preferred embodiment of the invention, the desired protein or polypeptide is surfactant protein B (SP-B) and variants thereof, such as Mini-B, Mini-B27, Mini-BLeu and KL4; Aβ, IAPP, β17; LL-37, hCAP18; surfactant protein C (SP-C) and variants thereof, such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu; surfactant protein A (SP-A) and variants thereof; and surfactant protein D (SP-D) and variants thereof; and Bri2-BRICHOS and Bri2. 113 - 231 Other preferred proteins according to the invention are nicastrin, neuroserpin, GFP, eGFP, and 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL and 0 LLLL proteins.

[0084] In certain preferred embodiments of the invention, the fusion protein is selected from the group consisting of SEQ ID NOs: 56, 60, 64, 66, 70-75, 82, 84, 87, 90 and 93; and proteins having at least 80%, preferably at least 90%, more preferably at least 95% identity to any of these proteins.

[0085] According to one aspect, the present invention provides a composition comprising an aqueous solution of the protein according to the present invention. In a preferred embodiment, the composition comprises an aqueous solution of the protein according to the present invention. The protein is preferably a fusion protein according to the present invention. The pH of the composition is preferably 7.2 or less, for example, 5.5 to 7.2.

[0086] In another aspect, the present invention provides an isolated nucleic acid, preferably a cDNA, encoding a protein according to the present invention. In a preferred embodiment, the isolated nucleic acid is selected from the group consisting of SEQ ID NOs: 58, 62, 65, 68, 76-81, 83, 85, 89, 91 and 95.

[0087] According to one aspect, the present invention provides a novel use of at least one portion of 100-160 amino acid residues having at least 70% identity to SEQ ID NO:1, wherein the amino acid residue corresponding to position 40 of SEQ ID NO:1 is selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 of SEQ ID NO:1 is selected from the group consisting of Asp and Glu, as part of a fusion protein, to improve the solubility of another part of the fusion protein, a desired protein or polypeptide as described herein. Preferred features of the solubility-improving moiety of the present invention are presented herein above.

[0088] In one preferred embodiment, the solubility enhancing moiety is used for the production of a desired protein or polypeptide, hi another preferred embodiment, the solubility enhancing moiety is used for studying or characterizing a desired protein or polypeptide.

[0089] An advantageous use of the moieties of the invention is as a solubility enhancing moiety in fusion proteins subjected to a pH below 7.2, for example a pH between 5.5 and 7.2. This particular variant of the N-terminal (NT) fragment of spider silk protein exists as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wild type NT forms dimers in the pH interval between 5.5 and 7.2, which increases the risk of undesired aggregation of the fusion protein. This is a pH interval useful for the functionality and stability of certain desired proteins and polypeptides. It is also a pH interval useful for certain purification protocols, for example when using ion exchange, such as cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) as the purification principle. It is also a pH interval useful for certain expression hosts, for example yeast.

[0090] According to another aspect, the invention provides a method for producing a desired protein or polypeptide. The first step comprises expressing in a suitable host a fusion protein according to the invention comprising the desired protein or polypeptide. The solubility enhancing moiety has at least 70% identity, such as 75%, 80%, 85%, 90%, 95% or even 100% identity, with any one of the charge-reversed NT proteins set forth in SEQ ID NO: 1-7 or charge-reversed variants of SEQ ID NO: 96, preferably SEQ ID NO: 1-2 or charge-reversed variants of SEQ ID NO: 96. As described herein, the amino acid residue corresponding to position 40 of SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; the amino acid residue corresponding to position 65 of SEQ ID NO: 1 is selected from the group consisting of Asp and Glu. In an alternative embodiment, the solubility enhancing moiety has at least 70% identity, such as 75%, 80%, 85%, 90%, 95% or even 100% identity, with the wild-type NT protein set forth in SEQ ID NO: 9 or 96. For the avoidance of doubt, this means that the method is also applicable to solubility-enhancing moieties that are not charge-reversed NT proteins, for example those not set out in SEQ ID NOs: 1-7.

[0091] Suitable expression hosts are well known to those skilled in the art and include, for example, bacteria and eukaryotic cells, such as yeast, insect cell lines and mammalian cell lines. Typically, this step involves the expression of the nucleic acid molecule encoding the fusion protein in E. coli.

[0092] The second method step involves obtaining a mixture containing the fusion protein and, optionally, isolating the fusion protein. The mixture may be obtained, for example, by lysing or mechanically disrupting the host cells. The mixture may also be obtained by collecting the cell culture medium if the fusion protein is secreted from the host. The resulting protein may be isolated using standard procedures. If desired, the mixture may be subjected to centrifugation and the appropriate fraction (precipitate or supernatant) is collected. The mixture containing the fusion protein may also be subjected to gel filtration, chromatography, such as ion exchange chromatography, for example cation or anion exchange chromatography, dialysis, phase separation or filtration to effect separation.

[0093] In a preferred embodiment, the resulting mixture contains the fusion protein dissolved in a liquid medium, typically a salt buffer or cell culture medium. This particular variant of the N-terminal (NT) fragment of spider silk protein exists as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wild-type NT forms dimers in the pH interval of 5.5-7.2, which increases the risk of undesired aggregation of the fusion protein. This is a pH interval useful for the functionality and stability of certain desired proteins and polypeptides, such as proteins / polypeptides prone to amyloid formation or aggregation. It is also a pH interval useful for certain expression hosts, such as yeast. It is also a pH interval useful when using certain purification protocols, such as ion exchange, e.g. cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) as the purification principle. In a preferred embodiment, this step further comprises purification of the fusion protein on an ion exchange medium, e.g. cation or anion exchange medium. In a preferred embodiment, this step further comprises purification of the fusion protein on an IMAC medium, preferably by elution with low pH (below the pKa of His, typically a pH of about 6).

[0094] Thus, the fusion protein is typically obtained as a solution in a liquid medium. The terms "soluble" and "in solution" mean that the fusion protein is not visibly aggregated and does not precipitate from the solvent at 60000 x g. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as 10-50 mM Tris-HCl buffer or phosphate buffer.

[0095] It has been advantageously found that the presence of the solubility enhancing moieties according to the invention improves the stability of the desired protein / polypeptide and prevents partial dimerization under these conditions. This can be advantageous when immediate polymerization is undesirable, for example during protein purification or in the preparation of large batches. In particular, this is advantageous for the method according to the invention, which comprises at least one step comprising subjecting the fusion protein to a pH below 7.2, for example a pH between 5.5 and 7.2. As mentioned above, this particular variant of the N-terminal (NT) fragment of spider silk protein exists as a soluble monomer, regardless of the pH of the surrounding aqueous medium. Wild type NT forms dimers in the pH interval between 5.5 and 7.2, which increases the risk of undesired aggregation of the fusion protein. This is a pH interval useful for the functionality and stability of certain desired proteins and polypeptides, for example proteins / polypeptides prone to amyloid formation or aggregation. It is also a pH interval useful for certain expression hosts, for example yeast.

[0096] In a preferred embodiment, the fusion protein is isolated from the mixture without a separation step, including gel filtration, chromatography or any other solid phase adsorption based separation. In a preferred embodiment, the fusion protein is isolated from the mixture by precipitation of the fusion protein, followed by suspending the precipitated fusion protein in an aqueous solvent, where the fusion protein is soluble in the aqueous solvent.

[0097] Precipitation of the fusion protein may be performed by any suitable technique that reduces the solubility of the fusion protein in the solvent, including changes in ionic strength and the addition of miscible organic solvents. Precipitation of the fusion protein is preferably achieved by salting out at high salt concentrations, i.e., subjecting the fusion protein to a sufficiently high salt concentration to render the fusion protein insoluble. By way of example, a NaCl concentration of 0.5 M or more, e.g. 1 M or more, is typically sufficient to precipitate the fusion protein. The precipitate containing the fusion protein is recovered, e.g., by filtration or centrifugation, and the filtrate or supernatant is discarded. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, the isolated precipitated fusion protein may be suspended and dissolved in a suitable solvent. Preferably, the precipitated fusion protein is suspended in an aqueous solvent in which the fusion protein is soluble.

[0098] In certain embodiments, the method further comprises the step of cleaving the fusion protein to separate the desired protein or polypeptide from the remaining solubility enhancing moiety or fragment thereof as a cleavage product; and optionally isolating the desired protein or polypeptide. If desired, the linker peptide can be removed by cleavage in this step. In these embodiments, the fusion protein comprises at least one cleavage site located between the at least one desired protein or polypeptide moiety and the at least one solubility enhancing moiety. In a typical fusion protein, this means the presence of a single cleavage site between the solubility enhancing moiety or moieties and the desired protein or polypeptide. Cleavage can be achieved using standard procedures, such as cyanogen bromide (CNBr) cleavage after the Met residue, hydroxylamine cleavage between the Asn and Gly residues, protease 3C cleavage at the -XLETLFQGX- site between the Gln and Gly residues, and a variety of other protease sites well known to those skilled in the art.

[0099] The resulting desired protein or polypeptide can be isolated using standard procedures. If desired, the mixture can also be subjected to centrifugation and the appropriate fraction (precipitate or supernatant) is collected. The mixture containing the desired protein or polypeptide can also be subjected to gel filtration, chromatography, dialysis, phase separation or filtration to effect separation. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, the linker peptide can be removed by cleavage in this step.

[0100] In a preferred embodiment, the desired protein or polypeptide is isolated from the mixture without a separation step, including gel filtration, chromatography or any other solid phase adsorption based separation. In a preferred embodiment, the desired protein or polypeptide is isolated from the mixture by extraction in an organic solvent, i.e., by extracting the desired protein or polypeptide by suspending the cleavage product in an organic solvent in which the desired protein or polypeptide is soluble and in which the residual solubility enhancing moiety or fragment thereof according to the invention is not soluble.

[0101] The organic solvents used according to the present invention are carbon-containing solvents and may exhibit various degrees of polarity. Although referred to as "solvents", it should be understood that these organic solvents are utilized to balance and alter the solubility of the fusion protein and / or the desired protein or polypeptide and / or the residual solubility enhancing moiety or fragment thereof during the production process. The protein or polypeptide may be very soluble in the organic solvent at a particular organic solvent concentration interval, but will drop and form a precipitate when the organic solvent concentration is increased or decreased. For example, the residual solubility enhancing moiety or fragment thereof may be soluble in a 50 / 50 (vol / vol) mixture of organic solvents, such as lower alkyl alcohol and water, but will drop and form a precipitate in a 90 / 10 or 10 / 90 (vol / vol) mixture. When subjected to non-precipitation conditions, such as a 50 / 50 or 0 / 100 mixture, the protein or polypeptide returns to a non-precipitating dissolved state. Those skilled in the art are well aware that other factors, such as temperature, pH, ionic strength, and type of organic solvent(s), may affect the critical organic solvent(s) concentration for precipitation of the protein or polypeptide. The threshold concentration for precipitation of a protein or polypeptide under given conditions is well known or can be readily determined by one of skill in the art.

[0102] The organic solvent according to the present invention may be selected from the group consisting of, but not limited to, pentane, hexane, cyclohexane, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, ethyl acetate, acetamide, diethyl ether, tetrahydrofuran, acetonitrile, methyl ethyl ketone, acetone, lower alkyl alcohols such as methanol, ethanol, propanol, isopropanol, butanol, or any mixture of the listed solvents. The organic solvent according to the present invention is preferably water-soluble. A preferred group of organic solvents are lower alkyl alcohols. The term lower alkyl alcohols refers to primary, secondary and tertiary alkyl alcohols having 1 to 6 carbon atoms, i.e. C 1-6Including alkyl alcohols. Specific examples of lower alkyl alcohols include methanol, ethanol, denatured spirits, n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Preferred lower alkyl alcohols are methanol, ethanol, isopropanol, and isobutanol, especially ethanol, due to its price, availability, and ease of handling.

[0103] In a preferred embodiment, the method further comprises a step of precipitation of the cleavage products prior to the extraction step. Precipitation of the cleavage products may be performed by any suitable technique that reduces the solubility of the desired protein or polypeptide in the solvent, including changes in ionic strength and the addition of miscible organic solvents. Precipitation of the cleavage products is preferably achieved by salting out at high salt concentrations, i.e., subjecting the cleavage products to a salt concentration high enough to render the desired protein or polypeptide insoluble. By way of example, a concentration of NaCl of 0.5 M or more, e.g. 1 M or more, is typically sufficient to precipitate the desired protein or polypeptide. The precipitate containing the desired protein or polypeptide is recovered, e.g., by filtration or centrifugation, and the filtrate or supernatant is discarded. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, the isolated precipitated desired protein or polypeptide can be suspended and dissolved in a suitable solvent.

[0104] Thus, a preferred method for producing a desired protein or polypeptide comprises the steps of: a) expressing in a suitable host a fusion protein of the invention comprising a desired protein or polypeptide; and b) obtaining a mixture containing the fusion protein, and b1) isolating the fusion protein, wherein this step comprises the steps of: b1a) precipitation of the fusion protein, preferably by salting out at high salt concentrations; and b1b) suspending the precipitated fusion protein in an aqueous solvent, where the fusion protein is soluble in the aqueous solvent; c) cleaving the fusion protein to release the desired protein or polypeptide from the remaining solubility-enhancing moiety or fragment thereof as a cleavage product; and c1) isolating the desired protein or polypeptide, wherein this step comprises the steps of: c1a) precipitation of the cleavage products; preferably by salting out at high salt concentrations; c1b) extracting the desired protein or polypeptide by suspending the cleavage product in a precipitating organic solvent, preferably comprising a lower alkyl alcohol, such as methanol, ethanol, or isopropanol; wherein the desired protein or polypeptide is soluble in the organic solvent; and wherein the residual solubility-enhancing moiety or fragment thereof is not soluble in the organic solvent. Contains:

[0105] The invention will now be further illustrated by the following non-limiting examples. EXAMPLES

[0106] Working Example Example 1 - Expression of NT and charge-reversed NT mutants NT wt (SEQ ID NO: 12 encoding SEQ ID NO: 11) and NT D40K / K65D The construct carrying (SEQ ID NO:8 encoding SEQ ID NO:2) was cloned into a pT7 expression vector and chemically transformed into competent E. coli BL21(DE3) cells. Plasmid-containing cells were inoculated into 10 mL of Luria-Bertani (LB) medium containing 70 mg / L kanamycin and grown overnight at 37°C and 180 rpm. 5 mL of the overnight culture was inoculated into 500 mL of LB medium (1 / 100) containing kanamycin and the cells were further incubated at 30°C until an OD of ∼1. 600The cells were grown to 30 °C for 1 h. Cells were induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and expression was carried out overnight at 20 °C. The next day, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8, to 30 mL, and stored at -20 °C for at least 24 h.

[0107] Example 2 -NT wt NT compared to D40K / K65D Biophysical properties of The NT dimerization process is highly dependent on intermolecular electrostatic interactions between residues D40 and K65, which play a key role in the initial association of monomers. In this study, we demonstrated that the dimerization of wild-type NT (NT wt 11), in which these residues are swapped. D40K / K65D ; SEQ ID NO:2) were designed and evaluated. Key biophysical properties were evaluated to determine the applicability of the mutants as solubility-enhancing fusion partners.

[0108] (A) Tryptophan fluorescence measurement NT D40K / K65D (SEQ ID NO: 2) and NT wt Fluorescence emission spectra for each of (SEQ ID NO: 11) were measured in a spectrofluorometer (Tecan Safire 2) using a Costar® black polystyrene assay plate with 96 flat-bottom wells. Proteins were diluted to a concentration of 5 μM in 20 mM HEPES / 20 mM MES adjusted to pH 5.6-8.0 in 0.4 pH unit steps. After excitation of the samples at 280 nm (5 nm bandwidth), emission spectra were recorded between 300-400 nm (10 nm bandwidth) in 1 nm steps. Tryptophan fluorescence ratios were calculated from the intensities at 339 nm and 351 nm and plotted as a function of pH. NT wt The data obtained for were fitted to a two-state binding model with a sigmoidal behavior of the monomer-dimer equilibrium.

[0109] The pH-dependent monomer-dimer equilibrium of NT can be monitored via the fluorescence shift of a single tryptophan (Trp) residue that is more exposed in the dimer. Figure 2 is a graph showing the monomer-dimer equilibrium measured by Trp fluorescence. Fluorescence spectra between 300 and 400 nm were measured in a buffer of 20 mM HEPES / 20 mM MES, and the ratio at 339 / 351 nm (wavelengths corresponding to the monomer and dimer conformations, respectively) was calculated to obtain the pH-dependent monomer-dimer equilibrium of NT. wt (dotted line) and NT D40K / K65D The ratio of fluorescence at 339 and 351 nm as a function of pH is plotted for NT with a dimerization pKa of pH 6.5 (filled line). wt The sigmoid plot for the fluorescent shift is shown in Fig. 1. D40K / K65D was not observed and ratios corresponding to the monomer were measured over the entire pH range (Figure 2).

[0110] (B) 15 N, 1 H]-HSQC NMR measurement NT mutants D40K / K65D (SEQ ID NO: 2) and NT wt Cells expressing (SEQ ID NO: 11) were cultured overnight and further 15 The cells were inoculated into 1 / 100-500 mL of minimal medium M9 containing N-labeled ammonium chloride and 70 mg / L kanamycin. The cells were incubated overnight at 22 °C until an OD of 1.4. 600 The cells were grown to 300 ug / ml. Proteins were expressed and purified as described above.

[0111] [ 15 N, 1 H]-HSQC NMR spectra were acquired at 25 °C on a Varian Unity Inova 600-MHz NMR spectrometer equipped with an HCN cold probe. 15 NMR samples of N-labeled proteins were prepared in either 20 mM sodium phosphate, 20 mM NaCl, pH 5.5, or 20 mM sodium phosphate, 300 mM NaCl, pH 7.2 buffer and analyzed by 2D [15 N, 1 H]- HSQC NMR spectra were recorded and analyzed using Bruker Topspin 3.1 software.

[0112] HSQC NMR, NT wt The measurements at pH 7.2 and 5.5, where NT is a monomer or dimer, respectively, showed large chemical shift differences. wt and N.T. D40K / K65D of 15 N- 1 Overlays of H HSQC NMR spectra were generated at pH 5.5 and at pH 7.2 (not shown). Figure 3 shows the NT at pH 5.5 (lower panel) and pH 7.2 (upper panel). wt and N.T. D40K / K65D are the average backbone amide 1H and 15N chemical shift differences between

[0113]

number

[0114] NT D40K / K65D The spectra obtained from are similar at both pH values, indicating that monomeric NT at pH 7.2 wt The spectrum corresponds to

[0115] (C) Urea-induced denaturation as a measure of stability NT wt (SEQ ID NO: 11) and NT D40K / K65D (SEQ ID NO:2) protein was diluted to 5 μM in 20 mM HEPES / 20 mM MES supplemented with urea from 0 to 7 M in 0.5 M steps. Protein stability at each concentration of urea was monitored by Trp fluorescence at constant pH values ​​ranging from 5.0 to 7.5 in 0.5 unit steps. For each measured pH, the fluorescence ratio was plotted against urea concentration and fitted to a two-state unfolding model to determine the transition point. NT wt (dotted line) and NT D40K / K65DThe data for (black line) are shown as the transition point between the native and denatured states as a function of pH in FIG. 4 ([den] 50% ).

[0116] Referring to Figure 4, NT wt is significantly more stable in the dimeric conformation at low pH. In contrast, NT D40K / K65D NT wt Similar to the dimer, and independent of pH changes, it shows increased overall stability over the entire pH interval.

[0117] (D) Temperature scan as a measure of stability. Temperature scans were performed and analyzed using circular dichroism (CD). Experiments were performed on a 410-model circular CD spectrometer (Aviv biomedical Inc., Lakewood, NJ, USA) using a 300 μL cuvette with a path length of 1 mm. For all measurements, NT wt (SEQ ID NO: 11) and NT D40K / K65D (SEQ ID NO:2) protein was diluted to 10 μM in 5 mM phosphate buffer at pH 5.5 or pH 8.0. CD spectra were recorded from 260 to 185 nm at 25°C, at 25°C after heating to 95°C and again at 25°C after cooling the sample. For each temperature, data are shown as the average of four scans. Temperature scans were measured at 222 nm by recording 1°C steps in the temperature interval from 25 to 95°C and the data were fitted to a two-state unfolding model.

[0118] Figure 5 shows the NT measured by CD spectroscopy. wt and N.T. D40K / K65D The CD signal at 222 nm was measured in m deg at pH 5.5 and pH 8.0, plotted as a function of temperature (°C), and fitted to a two-state unfolding model to determine the equilibrium melting temperature (T m The maximum melting temperatures were calculated using the NT at pH 8.0 and pH 5.5, respectively. D40K / K65D A significantly lower melting temperature was determined for NT at pH 8.0.wt Monomer, and NT at pH 5.5 wt was determined for the dimer.

[0119] Figure 6 shows the NT measured by CD spectroscopy. wt and N.T. D40K / K65D The refolding ability of the 100% 15 ... wt (A) at pH 8.0 and (B) at pH 5.5, and NT D40K / K65D was scanned between 185 and 260 nm at 25°C, 95°C, and after cooling, again at 25°C, at (C) pH 8.0 and (D) pH 5.5. Data are presented as smoothed averages of four measurements. CD spectra monitored before and after thermal denaturation show NT wt The results show that NT refolds into an alpha-helical structure, but the ellipticity decreases after refolding at pH 8 (Figure 6A), which is even more evident at pH 5.5 (Figure 6B). D40K / K65D 6C) and pH 5.5 (FIG. 6D), showing nearly identical alpha-helical spectra before and after temperature-induced unfolding.

[0120] Example 3 - Expression of fusion proteins NT respectively D40K / K65D and N.T. wt Fusion protein constructs carrying target peptides SP-C33Leu (SEQ ID NOs: 58-59 encoding SEQ ID NOs: 56-57) and KL4 (SEQ ID NOs: 62-63 encoding SEQ ID NOs: 60-61) in fusion with were cloned into pT7 expression vector and chemically transformed into competent E. coli BL21(DE3) cells. Fusion protein constructs carrying the same target peptides and proteins in fusion with PGB1 or Trx were subjected to the same procedure.

[0121] Plasmid-containing cells were inoculated into 10 mL of LB medium containing 70 mg / L kanamycin and grown overnight at 37°C and 180 rpm. 5 mL of the overnight culture was inoculated into 500 mL of LB medium (1 / 100) containing kanamycin and the cells were further incubated at 30°C to an OD of ∼1. 600 The cells were grown to 30 °C for 1 h. Cells were induced by addition of IPTG to a final concentration of 0.5 mM and expression was carried out overnight at 20 °C. The next day, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8, to 30 mL, and stored at -20 °C for at least 24 h.

[0122] Example 4 - Purification of fusion proteins for yield comparison NT mutants mediate solubility towards aggregation-prone fusion partners in comparison to the highly soluble PGB1 domain wt and N.T. D40K / K65D Trx was also evaluated in fusion with SP-C33Leu but was later excluded due to its poor performance.

[0123] The fusion protein obtained in Example 3 was solubilized by sonication in loading buffer (20 mM Tris-HCl, pH 8) at 80% amplitude, 1 sec on and 1 sec off, for a total of 3 min. Soluble and insoluble fractions were separated by centrifugation at 27000×g for 30 min at 4° C. The cleared lysate was loaded onto an IMAC column pre-packed with Ni-Sepharose (GE Healthcare) and equilibrated with loading buffer. Bound proteins were washed with 20 mM Tris-HCl, 5 mM imidazole, pH 8 and eluted in 1 mL fractions with 20 mM Tris-HCl, 300 mM imidazole, pH 8. The absorbance at 280 nm was measured for each fraction and protein-rich fractions were pooled. Imidazole was removed by dialysis overnight at 4° C. and 5 L of loading buffer using a Spectra / Por® dialysis membrane with a molecular weight cut-off of 6-8 kDa. Protein purity at each step was determined by SDS-PAGE using 15% acrylamide gels stained with Coomassie Brilliant Blue.

[0124] (A) SP-C33Leu fusion protein FIG. 7 shows SDS-PAGE evaluation of the SP-C33Leu fusion protein. wt (lane 1), NT D40K / K65D (lane 2), Trx (lane 3) or PGB1 (lane 4). Lane M shows size markers with molecular weights indicated to the left. Panel A shows expression analysis before induction and after overnight expression at 20° C. in Bl21 E. coli cells. Panel B shows solubility analysis after 3 min sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. Panel C shows fusion proteins after comparative Ni-Sepharose purification.

[0125] (B) KL4 fusion protein Figure 8 shows SDS-PAGE evaluation of the KL4 fusion protein.wt (lane 1), NT D40K / K65D (lane 2) or PGB1 (lane 3). Lane M shows size markers with molecular weights indicated to the left. Panel A shows expression analysis before induction and after overnight expression at 20° C. in Bl21 E. coli cells. Panel B shows solubility analysis after 3 min sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. Panel C shows the fusion proteins after comparative Ni-Sepharose purification.

[0126] In summary, the NT mutant NT wt , and N.T. D40K / K65D was abundantly expressed at similar levels in fusions with SP-C33Leu (Fig. 7A) and KL4 (Fig. 8A), and at higher levels compared to PGB1 and Trx. All proteins and peptides were expressed in the NT mutant NT wt , and N.T. D40K / K65D Trx-SP-C33Leu was found to be stable in fusion with Trx-SP-C33Leu, whereas Trx-SP-C33Leu showed signs of degradation during expression.

[0127] NT D40K / K65D Both NT and PGB1 were able to mediate high solubility for their target proteins / peptides, which were mostly found in the soluble fraction, with some minor differences (Figures 7-8B). wt showed ~50% soluble protein in fusion with KL4 (Figure 8B). The most striking difference between the NT mutants was observed in fusion with SP-C33Leu, where NT wt For , it mainly produced insoluble protein, and for NT D40K / K65D yielded a completely soluble protein (Fig. 7B).

[0128] NT on Ni-sepharose D40K / K65DPurification of the fusion proteins yielded 284 and 428 mg / L culture for SP-C33Leu and KL4, respectively (Figures 7-8C). This corresponds to a 2-8 fold higher amount compared to the proteins in fusion with PGB1, mainly due to the higher expression levels (Table 9). wt The yields for Trx were intermediate, approximately 1.3-4 fold higher compared to the PGB1 fusion protein. Trx in fusion with SP-C33Leu gave the lowest yield, and the fusion protein continued to degrade during purification (Figure 7C).

[0129] [Table 10]

[0130] Example 5 - Purification of SP-C33Leu and KL4 peptides Cells expressing the fusion protein obtained in Example 3 were lysed by sonication for 1.5 min at 80% amplitude, 1 sec on and 1 sec off, total time 3 min. Only during full-scale purification by precipitation, the sonication procedure was repeated after standing on ice for 5 min and the sample was centrifuged at 50000×g for 30 min. Sodium chloride was added to the supernatant to a final concentration of 1.2 M and the centrifugation was repeated. The pellet from the centrifugation was dissolved in 20 mM Tris-HCl, pH 8, and briefly sonicated for 1.5 min at 60% amplitude, 1 sec on and 1 sec off, 3 min to completely redissolve the fusion protein. CNBr cleavage was performed by adding 1.7 mL of 2 M HCl to 30 mL of lysis solution, followed by 1.7 mL of 1 M CNBr. The cleavage reaction was carried out overnight at room temperature. The next day, 800 mM sodium chloride was added to the cleavage reaction in a second precipitation step, then centrifuged at 20000×g for 30 min. The supernatant was removed and the pellet was dried at 37° C. and suspended in 99.9% ethanol. Insoluble material was removed by centrifugation at 20,000×g for 30 min.

[0131] (A) SP-C33Leu peptide (SEQ ID NO: 44) SP-C33LeuNT D40K / K65DIt was expressed as a fusion protein and purified in a process independent of the chromatography step. Figure 7D shows the NT protein synthesis using a NaCl precipitation / ethanol extraction protocol and CNBr bromide cleavage for removal of the fusion tag. D40K / K65D Figure 1 shows an SDS-PAGE evaluation of the purification of SP-C33Leu peptide from -SP-C33Leu, where P1, S1, P2, S2 and T represent the insoluble fraction, the soluble fraction, the pellet after the first precipitation, the supernatant after the first precipitation and the purified target peptide, respectively.

[0132] First, sonicated cell lysates were purified in one simple step by precipitating the majority of the fusion protein with 1.2 M sodium chloride, removing most contaminants (Figure 7D). The fusion protein was designed with a methionine residue located just N-terminal to the peptide, allowing for cleavage with cyanogen bromide (CNBr).

[0133] Following CNBr cleavage under acidic conditions, a second precipitation was performed with 0.8 M sodium chloride. Both SP-C33Leu and KL4 are soluble in organic solvents, such as ethanol, methanol, or isopropanol, and surprisingly, all NT-fragments generated by CNBr remain insoluble in these solvents. Thus, the precipitated pellet was further purified by suspension in 99.9% ethanol followed by centrifugation to isolate 20-30 mg / L culture of highly pure SP-C33Leu peptide in the soluble ethanol fraction (Figure 7D).

[0134] (B) KL4 peptide (SEQ ID NO: 46) NT D40K / K65D The procedure described above for purification of the SP-C33Leu peptide from -SP-C33Leu was D40K / K65D Purification of the KL4 peptide from NT-KL4 was also reproducible. Figure 8D shows the results of NT-KL4 purification using a NaCl precipitation / ethanol extraction protocol and CNBr bromide cleavage for removal of the fusion tag. D40K / K65DFigure 1 shows SDS-PAGE evaluation of the purification of KL4 peptide from -KL4, where P1, S1, P2, S2 and T represent the insoluble fraction, the soluble fraction, the pellet after the first precipitation, the supernatant after the first precipitation and the purified target peptide, respectively.

[0135] Ethanol extraction yielded 10-15 mg / L culture of pure KL4 peptide (Figure 8D).

[0136] Example 6 - ESI-MS characterization of SP-C33Leu Further characterization of purified SP-C33Leu (SEQ ID NO: 44) obtained in Example 5 by ESI-MS showed that the recombinantly produced peptide, dissolved in ethanol, had the correct covalent structure.

[0137] The ESI-MS spectrum of SP-C33Leu obtained in Example 5 is shown in Figure 9. The spectrum shown in panel A shows mainly monomeric SP-C33Leu with 3 or 4 charges and 1 or 2 sodium adducts, as well as a minor fraction of dimers with 7 charges. A small amount of a contaminant (approximately 4314.8 Da corresponding to the peak at 1079 m / z) could also be observed. Panel B shows the MS / MS spectrum of the m / z 1199.2 peak in panel A.

[0138] Example 7 - Effect of rSP-C33Leu on tidal volume and lung gas volumes The effects of SP-C and its derivatives on tidal volume and lung volume can be evaluated using an animal model with positive end-expiratory pressure (PEEP) (Almlen, A et al., Neonatology 92, 194-200 (2007)).

[0139] Premature newborn rabbits (gestational age 27 days) were treated at birth with 200 mg / kg of 2% rSP-C33Leu (SEQ ID NO: 44) in dipalmitoylphosphatidylcholine (DPPC) / palmitoyloleoyl-phosphatidylglycerol (POPG) 68:31 (w / w) at a concentration of 80 mg / ml. Animals receiving the same dose of Curosurf® served as positive controls and untreated littermates as negative controls. Animals were ventilated with a standard pressure sequence at 35 / 0 (peak-insufflation pressure [cm H2O] / positive end-expiratory pressure (PEEP) [cm H2O]) for 1 min, 23 / 3 for 15 min, 18 / 3 for 5 min, 13 / 3 for 5 min and 23 / 3 for 5 min. Finally, the lungs were ventilated with nitrogen at 23 / 3 cmH2O for an additional 5 min and then removed for gas volume measurements. Both tidal volumes and lung volumes are given as median values.

[0140] Figure 10 shows the effect of rSP-C33Leu on tidal volume. Tidal volumes during 30 min of ventilation are shown for immature newborn rabbits treated with 200 mg / kg of 2% rSP-C33Leu in DPPC:POPG (68:31 w / w) at a concentration of 80 mg / mL and compared to animals receiving the same dose of Curosurf, DPPC:POPG only (negative control), and untreated animals. Tidal volumes are significantly increased for animals treated with 2% rSP-C33Leu in DPPC:POPG (68:31) compared to untreated negative controls and controls treated with DPPC:POPG (68:31), approaching those obtained after treatment with Curosurf®.

[0141] Figure 11 shows the effect of rSP-C33Leu on lung volume. Lung gas volumes are shown for immature newborn rabbits treated at birth with 200 mg / kg of 2% rSP-C33Leu in DPPC:POPG (68:31 w / w) at a concentration of 80 mg / mL, and compared with animals receiving the same dose of Curosurf®, DPPC:POPG only (negative control), and untreated animals. Lung volumes for animals treated with 2% rSP-C33Leu in DPPC:POPG (68:31) and Curosurf are equal and significantly higher than those for animals treated with DPPC:POPG (68:31). Similar results were obtained with synthetic SP-C33Leu peptide in the same animal model (data not shown).

[0142] Example 8 -NT D40K / K65D Expression of β17 polypeptide using fusion proteins To broadly explore the amyloid aggregation mechanism, researchers engineered β17 (SEQ ID NO: 27), a polypeptide that is highly prone to aggregation and can form amyloid-like fibrils in vitro. β17 consists of six β-strands, each containing seven amino acids, separated by five short turns. The residues are arranged in a polar and non-polar pattern. β17 has previously been expressed with a myc-tag for immunodetection, resulting in the peptide being insoluble and having to be purified from inclusion bodies. Such purification processes require the use of denaturing conditions (8 M urea), are time-consuming, and result in an unstable protein. Also, studies of β17 fibril formation using Thioflavin T (ThT) as a reporter have suffered from premature aggregation.

[0143] NT D40K / K65D Constructs with β17 polypeptide in fusion with (SEQ ID NO:65 encoding SEQ ID NO:64) and PGB1 (control) were cloned and expressed according to Example 3. Figure 12 is an SDS-PAGE evaluation of the β17 fusion protein and the resulting purified polypeptide. The β17 polypeptide is a fusion protein that encodes the NT D40K / K65DNT was fused to the C-terminus of either NT (lane 1) or PGB1 (lane 2). Lane M shows size markers, with molecular weights indicated on the left. Panel A shows expression analysis before induction and after overnight expression at 20° C. in Bl21 E. coli cells. D40K / K65D The -β17 fusion protein was expressed in E. coli at abundant levels exceeding those observed for β17 in fusion with PGB1. Panel B shows a solubility analysis after 2 min sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. After 2 min sonication of harvested cells followed by centrifugation, both fusion proteins were found primarily in the soluble fraction.

[0144] Panel C shows the fusion proteins after comparative Ni-Sepharose purification. D40K / K65D For β17 in fusion with NT and PGB1, 228 and 92 mg / L cultures were obtained, which correlated with the intensity of the bands when analyzed by SDS-PAGE. D40K / K65D The solubility tag was removed by proteolysis of a thrombin recognition sequence located at the N-terminus of β17 followed by a second Ni-Sepharose purification step to separate the tag from the target protein. Panel D shows the NT-17A fusion protein purified using Ni-Sepharose chromatography and thrombin for cleavage and removal of the fusion tag. D40K / K65D Figure 2 shows purification of β17 (SEQ ID NO: 27) from -β17. Lanes represent the supernatant after sonication (S), flow-through (FT), purified fusion protein (F), thrombin cleavage (CL), and purified β17 target protein (T). The yield of soluble protein was 7.8 mg / L culture when measured immediately after purification.

[0145] In conclusion, NT as a solubility-enhancing fusion tag for β17 D40K / K65D allows for an efficient purification process under non-denaturing conditions.

[0146] Example 9 - Characterization of β17 Polypeptide (A) Gel filtration The hydrodynamic size of the β17 polypeptide (SEQ ID NO:27) obtained in Example 8 was characterized using gel filtration. Gel filtration was performed on a 24 mL Superdex-200 column flowing at 0.3 mL / min. Samples were injected using a 200 μL loop and TBS, 5 mM EDTA, pH 8, with or without 150 mM NaCl as running buffer. The column was calibrated with apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa), which eluted at 10.25, 12.54, 13.65 and 16.18 mL, respectively.

[0147] FIG. 13 shows the solubility of soluble NT in salt-free running buffer (FIG. 13A) and in running buffer supplemented with 150 mM NaCl (FIG. 13B). D40K / K65D Gel filtration of the -β17 fusion protein is shown. Analysis was performed after storage of the protein at -20° C. for 1 week (black line) or at 4° C. for several days (dotted line).

[0148] NT D40K / K65D The fusion protein remained highly soluble and migrated as a stable octamer after storage at -20°C for a week or at 4°C for several days when analyzed by gel filtration in the absence of salt (Figure 13A). Similar results were obtained in the presence of 154 mM NaCl and 1 mM EDTA, but under these conditions the protein migrated as a stable dimer (Figure 13B).

[0149] (B) ThT assay The aggregation kinetics was monitored using ThT fluorescence based on the enhanced quantum yield for ThT fluorescence upon binding to amyloid fibrils. 80 μM NT was incubated in microplate wells (Microplate Corning 3881, 96-well, low-binding, half-area, Corning Incorporated Life Sciences, Acton, MA) in 20 mM sodium phosphate buffer, pH 8.0, 0.2 mM EDTA, containing 10 μM ThT. D40K / K65DExperiments were performed with -β17. ThT fluorescence was recorded under static conditions at 37° C. using a Fluostar Omega or Optima plate reader (BMG Labtech, Offenburg, Germany) with an excitation filter of 440 nm and an emission filter of 480 nm.

[0150] Figure 14 shows β17 fibrillization after proteolysis of the fusion protein by thrombin. ThT fluorescence of the fusion protein in the presence of thrombin (1000:1) shows fibrillization of β17 indicated by increased ThT fluorescence over time (solid line). In the absence of thrombin, no fibrillization was observed for the fusion protein (dotted line). When purified in the absence of the fusion tag, β17 did not remain in solution long enough to study fibrillization by ThT assay (data not shown). Here, we show that the NT D40K / K65D They showed that this was feasible by keeping the protein in solution when fused to β17 and then proteolysis to release β17 at the start of the experiment.

[0151] Example 10 -NT D40K / K65D Expression of Bri2 BRICHOS domain using fusion proteins Bri2 is a TM glycoprotein consisting of an N-terminal region followed by a TM domain, a linker region, a BRICHOS domain and a C-terminal region. Although the function is relatively unknown, the protein has been linked to Alzheimer's disease, Aβ precursor protein processing, Aβ homeostasis, apoptosis, tumor suppression and male reproduction. Mutations in the Bri2 gene are associated with familial British dementia and familial Danish dementia caused by the accumulation of amyloid fibrils in the brain. The situation is similar to Alzheimer's disease (AD), which is characterized by the accumulation of amyloid beta peptides (Aβ), eventually forming plaques in the brain. The Bri2 BRICHOS domain is thought to act as an anti-amyloid chaperone and prevent amyloid formation, and is therefore of interest as a potential therapeutic target.

[0152] Truncated BRICHOS domain Bri2 113 - 231 ,NT D40K / K65D or NT wt (SEQ ID NOs:68-69, which encode SEQ ID NOs:66-67). Proteins were cloned and expressed in Bl21 or Origami E. coli cells essentially as per Example 3 at 20° C. overnight or at 30° C. for 4 hours.

[0153] Figure 15 is an SDS-PAGE assessment of expression and solubility of Bri2 BRICHOS fusion protein. 113 - 231 Domain, NT wt (lane 1) or NT D40K / K65D (lane 2). Lane M shows size markers, with molecular weights indicated to the left. Panel A shows expression analysis in E. coli cells before induction (lane B) and after expression (lane A) at 20°C overnight (left gel) or 30°C for 4 hours (right gel). Both fusion protein variants showed high expression levels in both bacterial strains, but NT wt was slightly more efficient in Origami cells. Panel B shows the solubility analysis after lysozyme treatment followed by separation of the soluble (lane S) and insoluble (lane P) fractions. Lysozyme treatment of harvested cells was inefficient, whereas NT D40K / K65D The fusion proteins were slightly more soluble. Panel C shows the solubility analysis after 2 min sonication in 20 mM Tris-HCl, pH 8, followed by separation of the soluble (lane S) and insoluble (lane P) fractions. After sonication, both fusion proteins were found mainly in the soluble fraction. The analysis was performed on cells previously expressed at 20° C. overnight (left gel) or at 30° C. for 4 h (right gel).

[0154] The fusion protein was purified with Ni-sepharose, followed by thrombin cleavage and a second purification step to remove the tag. 113 - 231Final yields of protein (SEQ ID NO:50) were 55 mg / L culture in Origami and 12 mg / L culture in Bl21 with any of the NT variants.

[0155] Example 11 - Characterization of Bri2-BRICHOS (A) SDS-PAGE analysis of purified Bri2-BRICHOS. Figure 16 is an SDS-PAGE analysis of the purification of Bri2-BRICHOS protein (SEQ ID NO: 50) obtained in Example 10. Samples from the purification step were subjected to NTLC analysis under reducing (Panel A) and non-reducing (Panel B) conditions. wt (lane 1) or NT D40K / K65D (lane 2) in fusion with Bri2 113 - 231 The fusion protein was analyzed for 100% fusion protein (F) and 100% fusion protein (W1-W4). Molecular weights are indicated on the left. The supernatant was loaded onto Ni-Sepharose and the flow-through was collected (FT) and followed by four washing steps with 10 mL of running buffer (W1-W4). The pure fusion protein was eluted with imidazole (F) and cleaved with thrombin (CL).

[0156] SDS-PAGE under reducing conditions (Figure 16A) showed three bands (15, 12 and 10 kDa) for both fusion protein variants that correlated well with the expected size of Bri2-BRICHOS (14 kDa) and NT (migrating as 12 kDa and smaller proteins). The 12 kDa band is most likely cleaved Bri2-BRICHOS obtained nonspecifically cleaved by thrombin. The same samples run under non-reducing conditions (Figure 16B) showed that the 15 kDa and 12 kDa bands were able to form the expected oligomers from Bri2-BRICHOS.

[0157] (B) Gel filtration The conformation of the Bri2-BRICHOS protein (SEQ ID NO: 50) obtained in Example 10 was characterized using gel filtration essentially as described in Example 9. Gel filtration was performed to estimate the proportion of the fusion proteins in their monomeric conformation.113 - 231 4 shows gel filtration to determine the oligomeric state of the fusion protein.

[0158] FIG. 17A shows the NT wt (dotted line) or NT D40K / K65D (black line) in fusion with Bri2 113 - 231 Gel filtration analysis of NT wt Analysis of -Bri2-BRICHOS allowed four peaks to be distinguished. The peak at 25 kDa was close to the expected size of the monomeric fusion protein (29.7 kDa) but was small compared to the other peaks. Dimeric (50 kDa peak) and trimeric (76 kDa peak) states were observed for both fusion proteins, and NT wt The fusion protein formed very large oligomers / aggregates (125 kDa peak). Interestingly, the amount of protein with a monomeric conformation (25 kDa peak) was significantly higher than that of the NT100 protein. wt Compared to fusion proteins, NT D40K / K65D The NT mutation increased the proper folding of Bri2-BRICHOS, leading to a larger fraction of soluble monomeric Bri2-BRICHOS.

[0159] FIG. 17B shows Bri2 without NT. 113 - 231 Gel filtration analysis was performed to compare Bri2-BRICHOS produced in fusion with the S-tag or produced alone, showing that purification of the NT D40K / K65D The oligomeric state of the -Bri2-BRICHOS fusion protein was determined. Without fusion to the NT mutant, the protein migrated close to the void volume and was either aggregated or in very large oligomers.

[0160] The above data is NT D40K / K65DThese results show that β-lactamase α-glycosylation mediates the solubility and correct folding of Bri2-BRICHOS and can prevent undesired protein oligomerization.

[0161] Example 12 -NT D40K / K65D Expression of SP-A and SP-D using fusion proteins SP-A and SP-D are essential soluble innate immune proteins of the lung that survey the lung and bind to pathogens, resulting in their neutralization, aggregation and clearance. They are also important regulators of the function of various immune cells and of the inflammatory immune response. Mice lacking either SP-A or SP-D show increased susceptibility to pathogen infections, including RSV, as well as an exaggerated inflammatory response after inoculation with infectious bacteria. In order to understand their molecular actions in the lung in models of human respiratory disease, significant efforts have been made to develop recombinant forms of SP-A and SP-D. Recombinant SP-A and SP-D may also have therapeutic potential for the treatment of various human lung diseases (Salgado, D. et al., Front Immunol 5, 623 (2014)).

[0162] Trimer oligomerization increases the affinity of SP-A and SP-D for carbohydrates on the surface of pathogens. Smaller fragments capable of forming trimers also show activity, but less efficiently than the native forms. Heterologous expression of full-length SP-A and SP-D has so far only been successful in mammalian systems, although truncated forms have been expressed in bacterial and yeast systems.

[0163] Full-length hSP-A1 (UniProt ID: Q8IWL2), hSP-A2 (UniProt ID: Q8IWL1) and hSP-D (UniProt ID: P35247) and their truncated fragments: hSP-A1 81 - 228 and hSP-A2 81 - 228 (Silveyra, P. & Floros, J., Gene 531: 126-132 (2013); SEQ ID NOs: 47-48), and hSP-D 204-355(Hakansson, K. et al., Structure 7: 255-264 (1999); SEQ ID NO: 49) was synthesized by the NT D40K / K65D (SEQ ID NOs:70-75 encoded by SEQ ID NOs:76-81). Fusion proteins were cloned and expressed in Bl21 or Origami E. coli cells essentially as per Example 3 at 20° C. overnight or at 30° C. for 4 hours.

[0164] Example 13 -NT D40K / K65D Expression of amyloid beta (Aβ) peptides using fusion proteins Aβ peptides are involved in the formation of amyloid plaques associated with Alzheimer's disease. The peptides are released from the amyloid precursor protein (APP) by proteolytic cleavage with beta and gamma secretases and can aggregate to form oligomers or larger fibrils that constitute amyloid plaques. The mechanisms of Aβ misfolding and fibrillation, as well as the in vitro and in vivo toxicity of fibrils and intermediate oligomeric states, have been extensively studied using synthetic peptides. 1-40 and Aβ 1-42 Successful recombinant production of peptides has been demonstrated by extraction from inclusion bodies under denaturing conditions, however this method does not allow control over the oligomeric state of the peptides and requires subsequent size-exclusion chromatography to obtain monomeric fractions prior to analysis.

[0165] To overcome this limitation, Aβ1-42 (Uniprot ID P05067) was D40K / K65D (SEQ ID NO:82 encoded by SEQ ID NO:83). The fusion protein is cloned and expressed essentially according to Example 3 in Bl21 or Origami E. coli cells at 20° C. overnight or at 30° C. for 4 hours.

[0166] Example 14 -NT D40K / K65D Expression of islet amyloid polypeptide (IAPP) using fusion proteins Islet amyloid polypeptide (amylin or IAPP; Uniprot ID P10997) is a peptide hormone that is co-secreted with insulin from pancreatic β-cells and plays an important role in regulating blood glucose levels. Studies suggest that pancreatic amyloid formed by IAPP is associated with the development of type II diabetes. IAPP is expressed as a propeptide called ProIAPP, which is processed to IAPP upon stimulation. Human proIAPP has previously been recombinantly expressed in fusion with a Trx solubility tag and purified by extraction from inclusion bodies under denaturing conditions. Recombinant human IAPP (hIAPP) peptides are available from a number of commercial sources and are delivered in fusion with solubility tags, such as GST, or conjugated to BSA or OVA to keep the peptide soluble.

[0167] Human IAPP, NT D40K / K65D (SEQ ID NO:84 encoded by SEQ ID NO:85). The fusion protein is cloned and expressed in Bl21 or Origami E. coli cells according to Example 3 at 20° C. overnight or at 30° C. for 4 hours.

[0168] Example 15 -NT D40K / K65D Expression of hCAP18 using a fusion protein Cathelicidins are a family of antimicrobial and endotoxin-binding proteins found in granules of vertebrate neutrophils. Members of this family share a highly conserved 12 kDa N-terminus known as the cathelin-like domain. The biologically functional domain resides at the C-terminus and is activated upon cleavage from the proprotein by serine proteases. The only human form of cathelicidin, hCAP18, is the proprotein for the antimicrobial peptide LL-37, which is released by extracellular cleavage by protease 3. hCAP18 inhibits the growth of Gram-negative bacteria with an efficiency comparable to that of the mature peptide LL-37. However, recombinant production of hCAP18 is associated with low yields and unknown solubility, therefore new strategies for recombinant production are needed.

[0169] hCAP18 without a signal peptide was wt or NT D40K / K65D (SEQ ID NOs:86-87, encoded by SEQ ID NOs:88-89). Both fusion proteins were expressed overnight at 20° C. in Origami E. coli cells. FIG. 18 shows the NT protein expression profile before induction (B) and after overnight expression at 20° C. (A). wt or NT D40K / K65D 1 shows an SDS-PAGE analysis of hCAP18 expressed in fusion with GFP. The arrow indicates the expected band corresponding to the protein of interest.

[0170] After sonication at 80% amplitude with 1 s pulses on and off for a total of 2 min with non-denaturing amounts of urea (2 M), 0.7% Tween or 10% glycerol as buffer additives, the fusion protein was found in the soluble fraction after centrifugation, independent of the buffer additives. The fusion protein was purified on Ni-sepharose with a final yield of approximately 50 mg / L culture.

[0171] Example 16 -NT D40K / K65D Expression of Nicastrin using a fusion protein The γ-secretase protein complex is a four-component protease that is involved in the processing of amyloid precursor protein (APP) and the generation of the Alzheimer's disease-associated peptide amyloid beta (Aβ). Modulation of the activity and specificity of γ-secretase represents a potential therapeutic strategy for the treatment of Alzheimer's disease. One of the components, nicastrin or NCT, is a type I transmembrane glycoprotein with a large extracellular domain (ECD), which is thought to play a key role in the recruitment of γ-secretase substrates. Heterologous E. coli production of full-length nicastrin ECD has not been reported previously.

[0172] The ECD of human nicastrin (UniProt ID: Q92542) was D40K / K65D(SEQ ID NO:90 encoded by SEQ ID NO:91). The fusion protein is cloned and expressed in Bl21 or Origami E. coli cells according to Example 3 at 20° C. overnight or at 30° C. for 4 hours.

[0173] Example 17 -NT D40K / K65D Expression of green fluorescent protein (GFP) using fusion proteins GFP exhibits bright green fluorescence when exposed to light ranging from blue to ultraviolet light. GFP is frequently used as a reporter of expression due to its relatively small size, which allows diffusion throughout the cell without interfering with biological processes. Many different mutants of GFP have been engineered, most importantly, the S65T mutation dramatically increased fluorescence and photostability. Enhanced GFP (eGFP) is the result of the S65T mutation plus the F64L point mutation, which showed increased folding efficiency at 37°C and enabled the practical use of GFP in mammalian cells.

[0174] eGFP wt and N.T. D40K / K65D The fusion proteins were cloned in fusion with GFP (SEQ ID NO:92-93, encoded by SEQ ID NO:94-95) and expressed overnight at 20°C in Bl21 E. coli cells. Cells were disrupted by lysozyme treatment instead of sonication. This method is less effective at solubilizing the protein, but was necessary to maintain GFP fluorescence that is otherwise partially lost during sonication treatment. Still, about 30-40% of the fusion protein was found in the soluble fraction after centrifugation, with the majority of the protein being recovered during Ni-sepharose purification.

[0175] Example 18 - NT in fusion with rSP-C33Leu D40K / K65D are arranged in micelle-like particles. Purified soluble NT D40K / K65D The -SP-C33Leu fusion protein (SEQ ID NO: 56) was obtained as described in Examples 3-4 and subjected to size exclusion chromatography (SEC) and transmission electron microscopy (TEM). For TEM, purified soluble NT D40K / K65DThe protein (SEQ ID NO:2) was used as a control.

[0176] Size Exclusion Chromatography The purified fusion protein was diluted to 2 mg / mL in running buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8.0). A Superdex 200 column was equilibrated with running buffer and 200 μL of the sample was passed through the column at a rate of 0.5 mL / min. Protein elution was detected by measuring the absorbance at 280 nm. Molecular weight standards ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) (GE Healthcare) were loaded and eluted at 8.56 mL, 10.65 mL, 12.06 mL, 12.96 mL, 14.26 mL and 15.64 mL, respectively.

[0177] Transmission electron microscopy Samples were diluted in 20 mM Tris, pH 8. For negative staining, 3 μl of sample was applied to glow-discharged carbon-coated copper grids, stained with 2% (w / v) uranyl acetate, and air-dried. Grids were checked using a JEOL JEM-2100f transmission electron microscope operated at 200 kV. Images were collected with a TVIPS TemCam-F415 4k x 4k CCD-camera (Tietz Video and Image Processing Systems GmbH, Gauting, Germany) using a normal magnification of 60000.

[0178] NT D40K / K65DAlthough the -SP-C33Leu monomer has a calculated molecular weight of 19 kDa, the SEC analysis shown in Figure 19 showed a distinct oligomeric population with an estimated size of 510 kDa, corresponding to particles with a hydrodynamic radius of approximately 10 nm. The presence of such micelle-like particles with a size of 10-15 nm was confirmed using negative staining transmission electron microscopy (TEM). In Figure 20, panel (a) shows a negatively stained NT D40K / K65D In panel (b), TEM of the -SP-C33Leu fusion protein shows particles of 10-15 nm size. D40K / K65D Protein TEM served as a negative control. D40K / K65D With the protein alone, no particles were observed.

[0179] Example 19 (Comparative) - NT wt and N.T. A72R Protein expression of a fusion protein comprising EP 2 644 619 A1 discloses the use of the solubility-enhancing moiety NT A72R and fusion proteins containing said portions. A72R is a constitutive monomer even below a pH of 6.4. A72R -SP-C33Leu (SEQ ID NO: 100) and NT wt -SP-C33Leu (SEQ ID NO: 57) was expressed in E. coli BL21(DE3) cells and purified according to Examples 3-4.

[0180] The resulting proteins were separated by SDS-PAGE and stained with Coomassie. The different fusion proteins were examined and their expression (production) levels per bacterium were found to follow the order: NT wt -SP-C33Leu > NT A72R -SP-C33Leu.

[0181] In conclusion, N.T. A72R NT wt did not improve fusion protein expression levels compared to

[0182] Example 20 - Expression and purification of modified spidroin proteins Modified fibronectin-derived RGD loop, FN cc The spidroin proteins with repeat portions and CT portions from the small ampullate spidroin (MiSp) were designated as NT D40K / K65D and Z (SEQ ID NOs: 101-102). The same fusion proteins, but with the CT portion from the major ampullate spidroin (MaSp), were also cloned (SEQ ID NOs: 103-104). To test the expression levels of the modified spidroin proteins, the fusion proteins were cloned into a pT7 expression vector and chemically transformed into competent E. coli BL21(DE3) cells.

[0183] Plasmid-containing cells were inoculated into 150 mL of LB medium containing 50 mg / L kanamycin and grown overnight at 30°C and 220 rpm. 5 mL of the overnight culture was inoculated into 500 mL of LB medium containing kanamycin (1 / 100) and cells were further grown at 30°C to an OD of ~1. Cells were induced by addition of IPTG to a final concentration of 0.3 mM and expression was carried out overnight at 15°C. After 19 hours of expression, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8, up to 40 mL, and stored at -20°C for at least 24 hours. Cells were solubilized by addition of lysozyme. Soluble and insoluble fractions were separated by centrifugation at 21612 x g for 30 minutes at 4°C.

[0184] O.D. 600Duplicate culture samples, equal to 1000×g, were taken 4 and 19 hours after induction during expression. Samples were pelleted by centrifugation at 13000×g at 4° C. for 10 min, the supernatant was discarded and the pellet was stored at −20° C. for at least 24 h. Pelleted material was lysed using CelLytic B™ with the addition of lysozyme. One sample per time point was used for analysis of soluble and insoluble fractions separated by centrifugation at 13000×g at RT for 10 min. The other sample was used for whole cell analysis without separation of soluble and insoluble fractions. Samples were added with an equal volume of reducing SDS-PAGE loading buffer, boiled at 95° C. for 5 min and subsequently loaded in equal amounts onto a 12% acrylamide gel. Proteins were transferred to membranes using the iBlot 2® Dry Blotting System. An iBind™ Western Device is used for blotting with IRDYE800CW® fluorophore-labeled chicken anti-his antibody. Detection is performed using a Liquor Odyssey fc® Imaging System.

[0185] The cleared lysate from the culture was supplemented with NaCl and imidazole to a final concentration of 500 mM and 20 mM, respectively, and loaded onto an IMAC column previously packed with Zn-Sepharose (GE Healthcare) and equilibrated with loading buffer (20 mM Tris, 20 mM imidazole and 500 mM NaCl, pH 8). Bound proteins were washed with 20 mM Tris-HCl, 49 mM imidazole, 500 mM NaCl, pH 8, and eluted in 1 mL fractions with 20 mM Tris-HCl, 220 mM imidazole, 500 mM NaCl, pH 8. The absorbance at 280 nm was measured for each fraction, and protein-rich fractions were pooled. Imidazole was removed by dialysis overnight at 4° C. and against 5 L of loading buffer using a Spectra / Por® dialysis membrane with a molecular weight cutoff of 6-8 kDa. Protein purity at each step was determined by SDS-PAGE using 12% acrylamide gels stained with Coomassie Brilliant Blue.

[0186] After storage at -20°C, the fusion protein was thawed at 4°C, centrifuged at 4570 x g for 10 min and concentrated. Samples were stored at 4°C. Stability was assessed numerically by measuring concentration over a 3-day span with Nano Dro™, as well as visually by SDS-PAGE using 12% acrylamide gels stained with InstantBlue™ protein stain. NT in maintaining the highly aggregation-prone purified spidroin protein in solution. D40 / K65D It can be concluded that is more effective than Z-tag.

[0187] Example 21 - Expression of spidroin fusion proteins To test the expression level of spidroin fusion proteins, the IgG fragment sCD40 covalently linked to 4RepCT was expressed in 100% PBS-C100 / 1000 ng / mL, respectively. D40K / K65Dand Z fusions were cloned into the pT7 expression vector (SEQ ID NOs: 105-106) and chemically transformed into competent E. coli BL21(DE3) cells.

[0188] Plasmid-containing cells were inoculated into 150 mL of LB medium containing 50 mg / L kanamycin and grown overnight at 30°C and 220 rpm. 5 mL of the overnight culture was inoculated into 500 mL of LB medium (1 / 100) containing kanamycin and cells were further grown at 30°C to an OD of ~1. Cells were induced by addition of IPTG to a final concentration of 0.3 mM and expression was carried out overnight at 15°C. After 19 hours of expression, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8, up to 40 mL, and stored at -20°C for at least 24 hours.

[0189] To analyze the amount of protein expressed in the soluble and insoluble fractions, respectively, culture samples were taken and analyzed by Western blot as described in Example 20.

[0190] Example 22 - Expression of IgG fragments NT respectively D40K / K65D The IgG fragment sCD40 covalently linked to a sortase recognition sequence in fusion with and Z (SEQ ID NOs: 107-108) was cloned into a pT7 expression vector and chemically transformed into competent E. coli BL21(DE3) cells.

[0191] Plasmid-containing cells were inoculated into 20 mL of LB medium containing 50 mg / L kanamycin and grown overnight at 30°C and 220 rpm. 5 mL of the overnight culture was inoculated into 500 mL of LB medium (1 / 100) containing kanamycin and cells were further grown at 30°C to an OD of ~1. Cells were induced by addition of IPTG to a final concentration of 0.3 mM and expression was carried out overnight at 15°C. After 19 hours of expression, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8, up to 40 mL, and stored at -20°C for at least 24 hours.

[0192] To analyze the amount of protein expressed in the soluble and insoluble fractions, respectively, culture samples were taken and analyzed by Western blot as described in Example 20.

Claims

1. 1. A fusion protein comprising: (i) at least one portion of 100-160 amino acid residues that is a solubility-enhancing moiety, said at least one portion having at least 90% identity to SEQ ID NO:1, wherein the amino acid residue at a position corresponding to position 40 of SEQ ID NO:1 is selected from the group consisting of Lys, Arg and His; and the amino acid residue at a position corresponding to position 65 of SEQ ID NO:1 is selected from the group consisting of Asp and Glu; and (ii) at least one moiety which is a desired protein or polypeptide; A fusion protein comprising:

2. The fusion protein according to claim 1 , wherein the amino acid residue at the position corresponding to position 40 of SEQ ID NO:1 is Lys.

3. The fusion protein according to claim 1 or 2, wherein the amino acid residue at the position corresponding to position 65 of SEQ ID NO: 1 is Asp.

4. below, (iii) at least one cleavage site disposed between the at least one desired protein or polypeptide moiety and the at least one solubility-enhancing moiety; The fusion protein of claim 1 , further comprising:

5. A method for producing a fusion protein comprising a desired protein or polypeptide, comprising the steps of: a) expressing in a suitable host a fusion protein according to any one of claims 1 to 4 comprising a desired protein or polypeptide; and b) obtaining a mixture containing the fusion protein; A method comprising:

6. A method for producing a desired protein or polypeptide comprising the steps of: a) expressing in a suitable host a fusion protein according to any one of claims 1 to 4 comprising a desired protein or polypeptide; b) obtaining a mixture containing the fusion protein; and c) cleaving the fusion protein to separate the desired protein or polypeptide from the remaining solubility-enhancing moiety or fragment thereof as a cleavage product; A method comprising:

7. Step c) comprises the steps of: c1) isolating the desired protein or polypeptide; The method of claim 6, comprising:

8. Step b) is the following step: b1) isolating the fusion protein; The method according to any one of claims 5 to 7, comprising:

9. 9. The method according to any one of claims 6 to 8, comprising the step b1) of isolating the fusion protein; and / or comprising the step c1) of isolating the desired protein or polypeptide.

10. 10. The method of claim 9, provided that the method does not include a separation step comprising gel filtration, chromatography or any other solid phase adsorption based separation.

11. Isolating the fusion protein, step b1), comprises the following steps: b1a) precipitation of the fusion protein; and b1b) suspending the precipitated fusion protein in an aqueous solvent, where the fusion protein is soluble in the aqueous solvent; The method according to any one of claims 8 to 10, comprising:

12. Isolating the desired protein or polypeptide, step c1), comprises the steps of: c1b) extracting the desired protein or polypeptide by suspending the cleavage product in an organic solvent; where the desired protein or polypeptide is soluble in the organic solvent; and where the residual solubility-enhancing moiety or fragment thereof is not soluble in the organic solvent.

12. The method of any one of claims 7 and 9 to 11, comprising:

13. 13. The method of claim 12, wherein the organic solvent in step c1b) comprises a lower alkyl alcohol, such as methanol, ethanol or isopropanol.

14. The step c1) of isolating the desired protein or polypeptide comprises, prior to the extraction step c1b), the following steps: c1a) Precipitation of cleavage products; 14. The method of any one of claims 12 to 13, further comprising:

15. 15. The method according to any one of claims 11 to 14, wherein at least one precipitation step comprises salting out at a high salt concentration.

16. 16. The method according to any one of claims 5 to 15, wherein the mixture obtained in step b) contains micelles of the fusion protein.

17. A micelle of a fusion protein according to any one of claims 1 to 4.

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