Incorporating unnatural amino acids using one pot pure system

Abstract

Disclosed are compositions and methods for incorporating non-naturally occurring amino acids into a polypeptide. This can be done by using the One Pot PURE (Protein Synthesis Using Recombinant Elements) system.

Description

[0001] INCORPORATING UNNATURAL AMINO ACIDS USING

[0002] ONE POT PURE SYSTEM

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of U.S. Provisional Application No. 63 / 515,219, filed July 24, 2023, herein incorporated herein by reference in its entirety.

[0005] STATEMENT ACKNOWLEDGING GOVERNMENT SUPPORT

[0006] This invention was made with government support under Grant No. W911NF-22-2- 0246 and Grant No. W91 INF-16-1-0372 awarded by the Army Research Office. The government has certain rights in the invention.

[0007] SEQUENCE LISTING STATEMENT

[0008] The specification further incorporates by reference the Sequence Listing submitted herewith via EFS. The Sequence Listing .xml file, identified as 10046-553WO1 ST26 is 30.9 KB and was created on July 24, 2024. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification, and does not contain new matter.

[0009] BACKGROUND

[0010] The incorporation of non-canonical amino acids into proteins has greatly expanded their functionalities,1,2including new-to-nature catalytic functions,3 5enhancement of preexisting properties,6,7and probing of enzymatic activities.8,9To this end, cell-free protein synthesis (CFPS) has emerged as an attractive means of readily introducing non-canonical amino acids. Eliminating membrane permeability as a barrier to substrates and reducing competition with endogenous messages has proven possible to greatly expand genetic code modifications.2,10,11In particular, the Protein synthesis Using Recombinant Elements (PURE) system relies on reconstituting individually purified transcription and translational factors,2"13and allows the ready programming of protein production through the simple introduction of template DNA. Relative to lysate-based methods, the PURE system contains less protease and nucleases activity, which greatly enhances translation, and leads to less batch-to-batch variation.11 Unfortunately, the PURE system suffers from high cost and / or requires intensive efforts to produce in a laboratory setting.14Some of these issues were addressed by developing a method in which individual overexpression strains for each of 36 translation and transcription factors are generated during a single co-culture, and purified jointly via a nickel chelate column.14

[0011] However, there is still a need for adapting the One Pot PURE system to incorporate non-canonical amino acids by introducing an engineered tRNA synthetase. These and other needs are at least partially addressed by this disclosure.

[0012] SUMMARY

[0013] The present disclosure is directed to a method of producing a polypeptide comprising at least one non-naturally occurring amino acid, wherein the polypeptide is produced using One Pot PURE (Protein Synthesis Using Recombinant Elements) system.

[0014] Yet in still further aspects, the disclosure is directed to a One Pot PURE system configured to produce a non-naturally occurring amino acid.

[0015] Yet in still further aspects, the disclosure is directed to a method of producing a polypeptide comprising at least one non-naturally occurring amino acid, wherein the method comprises: (a) coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask, wherein at least one of the protein-producing cells have been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis; (b) purifying the proteins produced in step a); (c) introducing to the proteins of step b) to components needed for introduction of a non-naturally occurring amino acid, thereby producing a polypeptide with a non-naturally occurring amino acid.

[0016] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description or can be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the chemical compositions, methods, and combinations thereof, particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. DESCRIPTION OF DRAWINGS

[0017] Figures 1A-1B illustrate an alternative protein expression strategies included changing into a more repressible plasmids (pKAR2) and changing into more compatible E. coli strain with genetically encoded repressors (BL21 Marionette) (FIG. 1A) and the activity of purified protein factor was assessed via in vitro transcription and translation of sfGFP reporter upon reconstitution (FIG. IB).

[0018] Figures 2A-2E illustrate an Overall schematic of producing customized One Pot protein factors for in vitro transcription and translation to synthesize wild-type GFP, or to incorporate unnatural amino acid into short peptide chain or into orange fluorescent protein depending on the input of different plasmid (FIG. 2A); formulas for unnatural amino acids used in one aspect of this disclosure: levodopa (L-DOPA) and N-E-propagyloxycarbonyl-L- lysine (ProgK) (FIG.2B);emission (left) and excitation (right) spectrum from the IVTT reaction indicating red-shift in Xrnax of orange fluorescent protein comparing to sfGFP (FIG. 2C); Peptide sequence containing in-frame ochre codon (UAA) and N-terminal Flag epitope (top); MALDI-MS spectrum (bottom) of ochre codon suppression for ProgK incorporation using modified One Pot protein factors (ARF1, +RF2) (FIG. 2D); and a schematic of sfGFP- N150TAA reporter for ProgK incorporation (top) and fluorescent data (ex / em: 480 / 512 nm) taken from IVTT reaction using modified One Pot protein factors (ARF1, +RF2) (FIG. 2E).

[0019] Figure 3 shows T5 promoter deletion in M15 strains with pQE30 / 60 plasmid system. The intact T5 promoter / operator is SEQ ID NO: 23. The sequencing result is SEQ ID NO: 24 (left side) and SEQ ID NO: 25 (right side).

[0020] Figure 4 shows MALDI-TOF MS spectrum of the translated peptide containing levodopa unnatural amino acid.

[0021] Figure 5 shows MALDI TOF-MS spectrum of purified Flag peptide containing an inframe opal codon from IVTT reactions using One Pot protein factor (ARF 2, +M. Alvus Pyrrolysyl aaRS). Calculated m / z of Flag-Trp: 1742.81 and Flag-ProgK: 1766.83.

[0022] Figure 6 shows MALDI-MS spectrum of purified Flag peptide containing an in-frame opal codon from IVTT reactions using One Pot protein factor (ARF 1, +M. Alvus aaRS). Calculated m / z of Flag-ProgK: 1766.83. Figure 7 shows cell-free protein synthesis of N-£-propagyloxycarbonyl-L-lysine containing sfGFP using modified One Pot protein factors containing 14, 1 / 8, and 1 / 16 volume ofRF 2.

[0023] Figures 8A-8C show a MALDI-MS report for opal codon (UGA) suppression to incorporate ProgK (FIG. 8A), and background (FIG. 8B- no ProgK) (FIG. 8C- no ProgK and no o-tRNA). Theoretical [M+H+] for Trp misincorporation and ProgK are respectively 1742.81 m / z and 1766.83 m / z with the peptide reporter as indicated

[0024] Figures 9A-9E show a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK (FIG. 9A- 1 / 1 RF2; FIG. 9B- U RF2; FIG. 9C- 1 / 4 RF2; FIG. 9D- 1 / 8 RF2; FIG. 9E- 1 / 16 RF2). Theoretical [M+H+] for ProgK is 1766.83 m / z; (*) indicated sodium adduct.

[0025] Figure 10 shows Mass Spectrometry spectrum of trypsin digested superfolded Green Fluorescent Protein (sfGFP) containing ProgK unnatural amino acid at 149th position. The relative m / z difference between residue 7th and 8th corresponds to ProgK insertion.

[0026] Figure 11 shows fluorescent data that confirm the incorporation of two unnatural amino acids, levodopa and ProgK, into an orange fluorescent protein (OFP) at 66th and 149th amino acid residue. Under full condition, the excitation and emission signals at red-shifted wavelength (535 nm / 585 nm vs. 480nm / 512nm) are more pronounced and unique to L-DOPA incorporation into OFP.

[0027] Figures 12A-D shows a) Expression plasmid of EF-P and its associated factors (EpmA, EpmB, and EpmC) was transformed into BL21(DE3) E. coli strain and introduced into One Pot PURE preparation; b) A sfGFP fusion reporter containing proline-rich motifs was used to assay the activity of EF-P in the cell-free protein synthesis reaction; c) Kinetic data monitoring the sfGFP fluorescent signals (ex / em: 480nm / 512nm) with One Pot PURE protein factor containing titrated amount of EF-P; d) End-point sfGFP fluorescent data extrapolated from kinetic data in part C.

[0028] DETAILED DESCRIPTION

[0029] The present invention can be understood more readily by reference to the following detailed description, examples, drawings, and claims, and their previous and following description. However, before the present articles, systems, and / or methods are disclosed and described, it is to be understood that this invention is not limited to the specific or exemplary aspects of articles, systems, and / or methods disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0030] The following description of the invention is provided as an enabling teaching of the invention in its best, currently known aspect. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various aspects of the invention described herein while still obtaining the beneficial results of the present invention. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the present invention without utilizing other features. Accordingly, those of ordinary skill in the pertinent art will recognize that many modifications and adaptations to the present invention are possible and may even be desirable in certain circumstances and are a part of the present invention. Thus, the following description is again provided as illustrative of the principles of the present invention and not in limitation thereof.

[0031] General Definitions

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0033] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

[0034] When a range is expressed, a further aspect includes from the one particular value and to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase "x to y" includes the range from 'x' to 'y ' as well as the range greater than 'x' and less than 'y'_ The range can also be expressed as an upper limit, e.g., 'x, y, z, or less' and should be interpreted to include the specific ranges of ‘x,’ ‘y,’ ‘z,’ 'about x,' 'about y,' and 'about z' as well as the ranges of 'less than x,' 'less than y, or 'less than z,' or 'less than about x,' 'less than about y, and 'less than about z.' Likewise, the phrase ' x, y, z, or greater' should be interpreted to include the specific ranges of ‘x,’ ‘y,’ ‘z,’ 'about x,' 'about y,' and 'about z' as well as the ranges of 'greater than x,' greater than y,' 'greater than z,' or 'greater than about x,' greater than about y,' 'greater than about z.' In addition, the phrase " 'x' to 'y'," where 'x' and 'y' are numerical values, also includes "about 'x' to about 'y'."

[0035] Such a range format is used for convenience and brevity and, thus, should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of " 0.1% to 5%" should be interpreted to include not only the explicitly recited values of 0.1% to 5% but also include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range.

[0036] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0037] In still further aspects, when the specific values are disclosed between two end values, it is understood that these end values can also be included.

[0038] In still further aspects, when the range is given, and exemplary values are provided, it is understood that any ranges can be formed between any exemplary values within the broadest range. For example, if individual numbers 1, 2, 3, 4, 5, 6, 7, etc. are disclosed, then the ranges 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, etc. are also disclosed. References in the specification and concluding claims to parts by weight of a particular element or component in a composition denote the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a mixture containing 2 parts by weight of component X and 5 parts by weight, components Y, X, and Y are present at a weight ratio of 2:5 and are present in such a ratio regardless of whether additional components are contained in the mixture.

[0039] A weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0040] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0041] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0042] As used herein, the terms "may," "optionally," “may optionally,” “can” and "can optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient, as well as cases in which the formulation does not include an excipient.

[0043] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. It is further understood that these phrases are used for explanatory purposes only. It is further understood that the term “exemplary,” as used herein, means “an example of’ and is not intended to convey an indication of a preferred or ideal aspect.

[0044] The expressions "ambient temperature" and "room temperature" as used herein are understood in the art and refer generally to a temperature from about 20 °C to about 35 °C.

[0045] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, or approximately occurs.

[0046] Still further, the term “substantially” can, in some aspects, refer to at least about 80 %, at least about 85 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or about 100 % of the stated property, component, composition, or other condition for which substantially is used to characterize or otherwise quantify an amount.

[0047] In other aspects, as used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition.

[0048] As used herein, the terms “substantially identical reference composition,” “substantially identical reference article,” or “substantially identical reference electrochemical cell” refer to a reference composition, article, or electrochemical cell comprising substantially identical components in the absence of an inventive component. In another exemplary aspect, the term "substantially," in, for example, the context "substantially identical reference composition," or “substantially identical reference article,” or “substantially identical reference electrochemical cell,” refers to a reference composition, article, or an electrochemical cell comprising substantially identical components and wherein an inventive component is substituted with a common in the art component.

[0049] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0050] By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0051] By “increase” or other forms of the word, such as “increasing,” is meant raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0052] “Detecting” is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting a nucleic acid molecule in sample. Detection can include a physical readout, such as fluorescence output.

[0053] The term “hybridization” is defined as forming base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule, for example. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).

[0054] An “isolated” biological component (such as a nucleic acid molecule) has been substantially separated, produced apart from, or purified away from other biological components. Nucleic acid molecules which have been “isolated” include nucleic acids molecules purified by standard purification methods, as well as those chemically synthesized. Isolated does not require absolute purity, and can include nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated. A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In a particular example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another particular example, a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid. Examples of modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA).

[0055] The major nucleotides of DNA are deoxyadenosine 5 '-triphosphate (dATP or A), deoxy guanosine 5 '-triphosphate (dGTP or G), deoxy cytidine 5 '-triphosphate (dCTP or C) and deoxythymidine 5 '-triphosphate (dTTP or T). The major nucleotides of RNA are adenosine 5 '-triphosphate (ATP or A), guanosine 5 '-triphosphate (GTP or G), cytidine 5 '-triphosphate (CTP or C) and uridine 5 '-triphosphate (UTP or U).

[0056] Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, as known in the art.

[0057] Examples of modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5 -methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyljuracil, and 2,6-diaminopurine.

[0058] Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof. The term “complementary binding” as used herein occurs when the base of one nucleic acid molecule forms a hydrogen bond to the base of another nucleic acid molecule. Normally, the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). For example, the sequence 5'-ATCG-3' of one ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA. In this example, the sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'. Nucleic acid molecules can be complementary to each other even without complete hydrogen-bonding of all bases of each molecule. For example, hybridization with a complementary nucleic acid sequence can occur under conditions of differing stringency in which a complement will bind at some but not all nucleotide positions.

[0059] Methods for preparing and using nucleic acid probes are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990).

[0060] The identity / similarity between two or more nucleic acid sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.

[0061] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0062] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38 A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0063] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166=1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15=20*100=75). One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.

[0064] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to the arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0065] The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the examples included therein and to the Figures and their previous and following description. General Description

[0066] The “PURE” system, which stands for Protein Synthesis Using Recombinant Elements” (Shimizu et al., The PURE system for protein production. Methods Mol Biol. 2014;1118:275-84), relies on reconstituting individually purified transcription and translational factors, and allows the ready programming of protein production through the simple introduction of template DNA. Except for the ribosomes and tRNAs, which are highly purified from E. coli. the PURE system reconstitutes the E. coli translation machinery with fully recombinant proteins.

[0067] The PURE system includes, but is not limited to:

[0068] His-Tagged Translation Factors and Energy regeneration system:

[0069] -Initiation Factors (IF1, IF2, IF3)

[0070] -Elongation Factors (EF-Tu, EF-Ts, EF-G) -Release Factors (RF1, RF2, RF3) -Ribosome Recycling Factor -20 Aminoacyl tRNA synthetases -Methionyl tRNA formyltransferase -Adenylate Kinase (AK-1), Creatine Kinase (CK), Nucleoside-diphosphate Kinase (NDK), and Prolyl Isomerase (PPiase)

[0071] E. coli Ribosomes

[0072] E. coli tRNAs

[0073] NTPs, Amino Acids, Salts, Buffer

[0074] In addition, recombinant T7 RNA polymerase can be used to couple transcription to translation. The PURE system represents an important step towards a totally defined in vitro transcription / translation system, thus avoiding the “black box” nature of the cell extractbased systems.

[0075] The PURE system can be used to express a wide range of protein targets and has the capacity for a yield of more than 100 pg / ml. The activity of the synthesized protein can often be directly assayed without purification due to the low background activity of the translation mixture. All recombinant protein factors inside the PURE system can be His- tagged, in some cases allowing the synthesized protein to be “reverse-purified” (New England Biolabs “Feature Article). Unfortunately, the PURE system suffers from high cost and / or requires intensive efforts to produce in a laboratory setting (US Patent 7,118,883, herein incorporated by reference in its entirety for its teaching concerning PURE). Previous reports have addressed these issues by developing a robust and low-cost method, termed One Pot PURE, in which individual overexpression strains for each of 36 translation and transcription factors are generated during a single co-culture, and purified jointly via a nickel chelate column, greatly reducing the labor, cost, and time for producing proteins in vitro at even higher yields than the canonical PURE system. Because of the inherent modularity of introducing translation factors, it is possible to adapt the One Pot PURE system for the incorporation of non- canonical amino acids through the simple expedient of introducing an engineered orthogonal aminoacyl tRNA synthetase into the co-culture. The basal incorporation and optimization of translation for two distinctive non-canonical amino acids (ncAA) via the suppression of amber and ochre stop codons is shown in Example 1.

[0076] An improvement upon the PURE system comes in the form of the “One Pot” PURE cell-free system, which is discussed in Lavickova et al. (ACS Synthetic Biology 2019 8 (2), 455-462) and in Grasemann et al. (OnePot PURE Cell-Free System. J. Vis. Exp. (172), e62625 (2021), both of which are incorporated by reference in their entirety for their teaching regarding OnePot PURE cell-free systems. In this system, protein purification is streamlined by coculturing and co-purification, which minimizes time and labor costs.

[0077] In the modified One Pot PURE system disclosed herein, two distinctive orthogonal aminoacyl tRNA synthetases (o-aaRS) were introduced to incorporate two distinctive unnatural amino acids. This system can go beyond the site-specific incorporation of one unnatural amino acid and enable the incorporation of multiple unnatural amino acids. To achieve this, multiple o-aaRS can be introduced at once in a highly modular and robust fashion, as compared to the traditional PURE system. With the advancement of bioinformatics, new and novel o-aaRS can be virtually introduced, screened, and generated at a large scale, along with other transcription and translation machineries required for cell- free protein synthesis, all within a relatively short time frame.

[0078] Disclosed herein are methods and compositions which improve upon the OnePot PURE system by allowing for the introduction of at least one non-naturally occurring amino acid into a polypeptide produced using One Pot PURE system. This system can be done in single container, such as a flask. In the systems and methods disclosed herein, at least one of the protein-producing cells are modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis. This system can use, for example, E. coli, and different clones can be used to produce the elements needed for protein synthesis to take place. In one example, between 30 and 40 E. coli clones are used. Specifically, 36 clones can be used. One of skill in the art can assess how many clones can be produced based on the protein to be produced and the protocol required to do so.

[0079] After the desired proteins have been produced, the protein can be purified. This can be done by a variety of ways known in the art, including, but not limited to, affinity tags using metal, such as single nickel nitrilotriacetic acid (Ni-NTA) purification.

[0080] The non-naturally occurring amino acid can be incorporated by orthogonal tRNA synthetase. Combinations of orthogonal tRNA synthetase-tRNA pairs which can be used with the present invention include, but are not limited to, those found in Cervettini, et al. Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairs. Nat Biotechnol 38, 989-999 (2020), herein incorporated by reference in its entirety for its teaching concerning tRNA / orthogonal tRNA synthetases. An example of specific non- naturally occurring amino acids which can be incorporated include, but are not limited to, levodopa (L-DOPA) and N-e-propagyloxycarbonyl-L-lysine (ProgK).

[0081] Examples of other non-naturally occurring amino acids include, but is not limited to, Examples of unnatural amino acids include, but are not limited, to a p-acetyl-L- phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p- propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl- GlcNAcpP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L- phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p- amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, 24ufa24hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a P- amino acid; a y-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[0082] Any number of non-natural amino acids can be incorporated into the proteins produced by the methods disclosed herein. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more can be incorporated into the same protein.

[0083] Codon suppression can be used to incorporate non-naturally occurring amino acids. Three of the 64 different triplet codons do not code for an amino acid, but cause recruitment of a release factor resulting in disengagement of the ribosome and termination of the synthesis of the growing polypeptide. These codons are called ochre (TAA), opal (TGA), and amber (TAG). Furthermore, stop codons can be suppressed.

[0084] One of the significant improvements over the prior art is that, in the present invention, at least one expression plasmid has been engineered so that all genes of the plasmid are under the control of the T7 RNA polymerase promoter. In another aspect, a TetR operator adjacent to the T7 RNA polymerase promoter is expressed.

[0085] In another aspect, a Tet repressor can be included on at least one expression plasmid.

[0086] In yet another aspect, plasmids encoding one or more translation factors can be transformed into an engineered bacterial strain, such as, for example, E. coli BL21 Marionette strain, which contains a genome-encoded array of 12 different, constitutively expressed repressors including Lacl. The BL21 Marionette strain was used to express Alanine aminoacyl tRNA synthetase (AlaRS), Asparagine aminoacyl tRNA synthetase (AsnRS), Phenylalanine aminoacyl tRNA synthetase (PheRS), Threonine aminoacyl tRNA synthetase (ThrRS), Initiation Factor-1,2, 3 (IF- 1 ,2,3), Elongation Factor G (EF-G), Elongation Factor Thermostable (EF-Ts), Release Factor-1,2,3 (RF-1,2,3), Ribosome Recylcing Factor (RRF), Creatine kinase (CK), Nucleoside diphosphate kinase (NDK), T7 RNA Polymerase (T7RNAP). Any two or more of these can be used together in the engineered strain. The Marionette strain is explained in detail in Meyer et al. (Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2): 196-204, hereby incorporated by reference for its teaching concerning Marionette strain). Also contemplated are other strains with properties similar to the BL21 Marionette strain, such as those which overexpress lad.

[0087] The methods disclosed herein can be used to determine how best to produce a protein comprising a certain non-naturally occurring amino acid. In other words, optimization studies can be done which allows a practitioner to determine the best method of producing the protein comprising the non-naturally occurring amino acid to maximize efficiency and accuracy.

[0088] Therefore, disclosed herein is a method of producing a polypeptide comprising at least one non-naturally occurring amino acid, wherein the method comprises: a) coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask, wherein at least one of the protein-producing cells have been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis; b) purifying the proteins produced in step a); and c) introducing to the proteins of step b) to components needed for introduction of a non-naturally occurring amino acid, thereby producing a polypeptide with a non-naturally occurring amino acid.

[0089] Production of at least one non-naturally occurring amino acid can be optimized using this method. For example, one or more clones can be introduced in different ratios during co-culturing, and the results can be compared to determine which has greater yield of non-naturally occurring amino acid. The results can be compared to a control. The control can comprise, for example, a protein produced without the non-naturally occurring amino acid, or can have other, various components of the system adjusted for comparison. In this case “yield” can comprise more insertions of non-naturally occurring amino acid in polypeptide, or more overall polypeptide with non-naturally occurring amino acids incorporated, as compared to a control.

[0090] EXAMPLES

[0091] Example 1: Changes in coding and efficiency through modular modifications to a One Pot PURE system for in vitro transcription & translation The incorporation of unnatural amino acids are attractive methods for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. As disclosed herein, the One Pot PURE system was modified for a robust and modular system of enzymatic single site-specific of unnatural amino acid. The flexibility of this system was demonstrated through the introduction of two orthogonal aminoacyl tRNA synthetases and the suppression of two distinctive stop codons.

[0092] Results and Discussion:

[0093] Modifying the One Pot PURE system for more stable expression:

[0094] Initial attempts to utilize the One Pot PURE system revealed batch-to-batch variability that could be traced to the mutation of T5 promoter elements driving the expression of transcription and translation factors (FIG. 3). Among the 16 components that utilized either the pQE30 or pQE60 backbones, a T5 promoter deletion (around the -10 region) was found in 12 plasmids following two weeks of storage as frozen glycerol cell stocks and subsequent outgrowth. This issue could in turn, be ascribed to the fact that the pQE30 and pQE60 series plasmids, upon which much of the system was built, do not themselves encode genetic repression of translation factor expression, and thus loss or variation in copy number of a second, Lacl-expressing plasmid (pREP4) may lead to undue metabolic stress on at least some of the strains during the growth of the system as a whole. This issue has also been noted by Pluckthun and Jia, separately15,16.

[0095] To overcome this deficiency, more firmly repressing the expression of transcription and translation factors was attempted. Two strategies were explored. First, all genes were expressed under the control of the T7 RNA polymerase promoter and an adjacent TetR operator and included the Tet repressor on each expression plasmid (the so-called pKAR2 series of vectors17, FIG. 1A). The pKAR2 plasmids were transformed into a B21(DE3) strain for expression. Without wishing to be bound by any theory, it is hypothesized that an additional advantage of this configuration is that the T7 RNA polymerase promoter in the genome is under the control of LacI, allowing for two levels of control for expression of any individual translation factor (via IPTG and tetracycline induction). Second, the original pQE30 and pQE60 plasmids encoding translation factors were transformed into an E. coli BL21 Marionette strain, which contains a genome-encoded array of 12 different, constitutively expressed repressors including Lad18(FIG. 1A). These reconfigured systems were compared with a pristine version of the original One Pot PURE system (Ml 5).

[0096] For each system, the 36 strains were grown together, co-purified translation factors, and carried out in vitro transcription and translation (IVTT) assays with independently purified ribosomes and using sfGFP as a reporter. Expression in the BL21 Marionette strain yielded an IVTT mixture as active as the original mixture, while T7 RNAP -based expression yielded a less active mixture, at least under the induction conditions utilized (FIG. IB).

[0097] Non-canonical amino acid incorporation in One Pot PURE via amber codon suppression:

[0098] One utility of the One Pot PURE system is for rapidly testing the abilities of orthogonal tRNA synthetase (aaRS):tRNA pairs for augmenting the genetic code. An orthogonal aaRS:tRNA pair capable of incorporating L-DOPA (FIG. 2B) via Amber codon suppression19, and a corresponding reporter for L-DOPA incorporation, an orange fluorescent protein (OFP) derivative (FIG. 2A)19were used. The OFP reporter contains mutations at S205C and in-frame Amber stop codon at the Y66 position, and only successful incorporation of L-DOPA yields a signal that is red-shifted in both excitation and emission maxima wavelength (ex / em: 518 / 526 nm) comparing to the wildtype sfGFP (ex / em:490 / 508 nm). Without L-DOPA, no signal is observed at the expected excitation and emission wavelength of OFP or sfGFP. An L-DOPA aaRS with a C-terminal histidine tag expression construct (via a BL21 (DE3) strain and the pKAR2 vector containing an inducible T7 RNAP promoter) was introduced into a One Pot culture, while the strain producing RF-1 (Release Factor- 1, that normally mediates termination at Amber codons) was excluded. The suppressor tRNA was prepared in parallel by in vitro transcription of a linear DNA template and purification via denaturing gel electrophoresis.

[0099] The reconstituted system that included both the orthogonal AARS and purified suppressor tRNA was prepared, and the in vitro transcription and translation of a small FLAG-tagged peptide containing an in-frame amber codon was initially carried out. MALDL TOF analysis indicated successful incorporation of L-DOPA with high fidelity (no tyrosine incorporation was observed; FIG. 4). Plasmids encoding either the OFP reporter with an Amber codon or a wild-type GFP reporter were also introduced into the reconstituted IVTT reaction that contained the DOPA aaRS. The excitation and emission spectrum following translation indicated a red shift in Xmax for the Amber-suppressed, DOPA-containing OFP compared to the wild-type GFP (FIG. 2C).

[0100] Capturing the next stop codon for efficient non-canonical amino acid incorporation:

[0101] To demonstrate the adaptability and programmability of the system, suppress the other two stop codons, opal (UGA) and ochre (UAA) was performed. This expansion can allow to enzymatically incorporate two distinct non-canonical amino acids using the One Pot PURE system. The suppression of the opal stop codon using an orthogonal pyrrolysyl aminoacyl- tRNA synthetase :tRN A pair was first examined20,21. Since the opal codon (UGA) is only recognized by the Release factor-2 (RF-2),22the cells expressing RF-2 were eliminated from the One Pot culture to prevent termination and inefficient protein production, while including cells expressing the orthogonal pyrrolysyl aminoacyl-tRNA synthetase (PylRS). This modular ability to delete and include factors at will is a major advantage of the One Pot system. The orthogonal aaRS variants from Methanosacrina Barkeri (M Barker!) and Methanomethylophilus Alvus (M Alvus) were tested for the incorporation of N-e- propagyloxycarbonyl-L-lysine (ProgK) (FIG. 2B). Then, the FLAG peptide was evaluated in vitro transcription and translation and suppression of an in-frame stop codon in a short epitope tag, (FIG. 5). Only the M. Alvus PylRS yielded the desired peptide product in the One Pot system (FIG. 5).

[0102] It was noted that there was a noticeable misincorporation of tryptophan across from the opal codon, even with the addition of purified orthogonal tRNAUCAand ProgK (FIG. 5). Without wishing to be bound by any theory, it was assumed that tryptophan misincorporation is due to wobble base-pairing, given the similarity of the tryptophan (UGG) and opal (UGA) codons, and had been observed in previous reports.27 29Thus, the suppression codon was changed to an ochre stop codon (UAA) and the anticodon of the orthogonal M. Alvus Pyl tRNA was changed to the ochre codon UUA (M. Alv tRNA, ,) to potentially increase discrimination. To demonstrate ProgK incorporation a sfGFP reporter that contained an inframe ochre stop codon at the 150th amino acid and in a final opal termination codon (sfGFP- N150TAA) was used. Ochre codons are suppressed by both RF1 and RF2 in roughly equal measure.30,31To improve suppression the RF1 was eliminated whileRF 2 was retained to ensure proper termination and help eliminate background incorporation of canonical amino acid.22A FLAG peptide product containing ProgK and no misincorporation of natural amino acids was observed (FIG. 2D) the suppression was improved by lowering the amount of RF 2,22while fidelity was retained, which can be accomplished in the One Pot system by decreasing the volume of cells expressing RF2, as a result. One Pot mixture containing U volume of RF 2 yielded higher amounts of active sfGFP protein with only a slight increase in background (FIG. 2E). Further dilution of RF 2 did not significantly improve background (FIG. 6) nor yield (FIG. 7).

[0103] FIG. 8A shows a MALDI-MS report for opal codon (UGA) suppression to incorporate ProgK. The MALDI-MS report (background) for opal codon (UGA) without ProgK is shown in FIG. 8B and without ProgK and no o-tRNA is shown in FIG.8C. Theoretical [M+H+] for Trp misincorporation and ProgK are respectively 1742.81 m / z and 1766.83 m / z with the peptide reporter as indicated.

[0104] FIG. 9A shows a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK at 1 / 1 RF2. FIG. 9B shows a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK at U RF2; FIG. 9C shows a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK at 1 / 4 RF2. FIG. 9D shows a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK at 1 / 8 RF2 and FIG. 9E shows a MALDI-MS report for ochre codon (UAA) suppression to incorporate ProgK at 1 / 16 RF2. Theoretical [M+H+] for ProgK is 1766.83 m / z; (*) indicated sodium adduct.

[0105] FIG. 10 shows Mass Spectrometry spectrum of trypsin digested superfolded Green Fluorescent Protein (sfGFP) containing ProgK unnatural amino acid at 149th position. The relative m / z difference between residue 7th and 8th corresponds to ProgK insertion.

[0106] FIG. 11 shows fluorescent data that confirm the incorporation of two unnatural amino acids, levodopa and ProgK, into an orange fluorescent protein (OFP) at the 66th and 149th amino acid residues. Under full condition, the excitation and emission signals at red-shifted wavelength (535 nm / 585 nm vs. 480nm / 512nm) are more pronounced and unique to L-DOPA incorporation into OFP.

[0107] Overall, the One Pot PURE system was adapted for the incorporation of non-canonical amino acids and the suppression of both an amber (UAG) and ochre (UAA) stop codon in a single mRNA was demonstrated. It was shown the advantages of One Pot system to allow customization that is not possible in PURE system.

[0108] Methods:

[0109] Materials: All chemicals and biologicals reagents were purchased without any further purification. LB medium was purchased from Fisher Sci. Carbenicillin antibiotic and Isopropyl P-D-l -thiogalactopyranoside (IPTG) were purchased from Golden Bio. Levodopa (L-DOPA), N-8-propargyloxycarbonyl-L-lysine (ProgK), and anhydrotetracycline (aTC) were purchased from Sigma- Aldrich. Ni-NTA resin, dialysis cassettes (3.5K MWCO), and gravity flow column were purchased from Thermofisher. Amicon Ultracentrifugal Filters (3K MWCO), Anti -Flag M2 agarose, ZipTip desalting pipette tip, and E. coli tRNA were purchased from Sigma- Aldrich. All buffers used are listed in Table 2 and 3. 2- Mercaptoethanol was added immediately before use.

[0110] Escherichia coli Strains, Plasmids, linear DNA template, and tRNA: E. coli BL21(DE3) strain (purchased from New England Biolab, NEB), BL21Marioentte (purchased from Addgene, cat. 108253), and M15 were used for protein expression. For cloning and propagating plasmids, DhlObMarionette (purchased from Addgene, cat. 108251) was used to maintain the integrity of the pQE-series plasmid.

[0111] Plasmid pJLl-sfGFP was purchased from Addgene (cat. 69496). All the expression plasmids to construct One Pot protein factors were purchased through Addgene (cat. 124103- 124138). Plasmids encoding M. Barkeri PyLRS and M. Alv PylRS pET BCS barkeri and pET-AlvusPylRS-CtermHIS were generously donated. pKAR2-Mj DOPARs was cloned using pKAR2-DO dropout plasmid and synthetic DNA gene blocks or gel-purified PCR product from amplifying pQEs and pETs series plasmids containing appropriate Bsal cut-site and overhang for Golden Gate cloning.

[0112] The DNA template used for in vitro transcription was prepared by overlap PCR extension from primers in Table 5. The PCR product was precipitated using phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation, suspended with nuclease-free water and directly used with Hi Scribe T7 RNA Polymerase kit (purchased from NEB). Full-length of transcribed product was purified via TBE:Urea PAGE gel, precipitated with ethanol, and resuspended with nuclease-free water. Quantification of tRNA was used via NanoDrop. DNA template encoding short peptide containing in-frame stop codon and FLAG epitope was prepared with overlap PCR extension from primers in Table 5. The PCR product was precipitated using phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation, suspended with nuclease-free water and directly used in CFPS.

[0113] Preparation of His-tag gravity column: Ni-NTA resin (2 mL) is added into gravity flow chromatography column. The resin was then equilibrated with buffer A (17.5 mL) and ready to use. Modified preparation for One Pot protein purification: Preparation for On ePot protein purification in this work was modified from previous report814. Cultures for protein expression were all inoculated in LB broth, supplemented with 100 pg / mL carbenicillin and 50 pg / mL Kanamycin (only when Ml 5 strain was used), and grown at 37 °C, 240 rpm. All overnight and saturated cultures were inoculated in 0.3 mL of LB in a 96 deep-well plates. The strain expression EF-Tu and orthogonal aaRS for unnatural amino acid incorporation were inoculated and grown in 3 mL of LB in a standard 14 mL culture tube. The composition of One Pot expression culture at 250 mL of LB media in a 500 mL flask followed Table 1. The expression culture was first grown for 2h before induction with 0.1 mM IPTG and 100 ng / mL (only when pKAR2 series plasmid was added in One Pot expression culture) for 3h. Cells were then harvested by centrifugation (4000 xg, 10 minutes, 4 °C) and stored at -80 °C overnight.

[0114] Frozen cells were allowed to thaw on ice for 5 minutes from -80 °C and resuspended in 10 mL of Buffer A. The suspended cell pellets were transferred into a 50 mL Falcon tube. Cells were lysed using the sonication method on ice with the addition of splashes of brine (10s ON / 10s OFF, 35% amplitude, -2000 kJ delivered or 2 minutes and 40 seconds). Lysed mixture was then transferred to Nalgene Oak Ridge centrifuge tube. Cell debris was removed by centrifugation (15,000 rpm, 20 minutes, 4 °C). Supernatant was then transferred to a prepared His-tag (as described above) gravity column and incubated for 3 hours at 4 °C with constant shaking. After incubation, unbound lysate was allowed to flow through the column. It was then washed with 25 mL of Wash Buffer (95% buffer A, 5% buffer B) and eluted with

[0115] 3 mL of Elution Buffer (10% buffer A, 90% buffer B). The purified lysate was then dialyzed using a 3500 MWCO (Molecular Weight Cut Off) dialysis cassette overnight in HT buffer at

[0116] 4 °C.

[0117] On the final day of purification, dialyzed solution was added on a 15 mL Amicon Ultra filter with 12.5 mL of HT buffer for the final buffer exchange. The solution was concentrated to -1 mL using a centrifuge at 4000 rpm at 4 °C. The protein solution was mixed with Storage Buffer B in an equivalent amount. Another buffer exchange was accomplished using a 0.5 mL Amicon Ultracentrifugal filter unit at 14,000 rpm at 4 °C for 45 minutes. A purified protein mixture was collected and quantified using Bradford Assay with bovine gamma globulin as standard (detailed description in the subsequent section). The working protein factor was diluted or concentrated to 12,250 ng / mL and stored at -80 °C for further usage. Ribosome purification: E. coli Al 9 strain was grown overnight in 100 mL of LB broth overnight at 37 °C, 270 rpm. On the next day, 30 mL of overnight culture was inoculated in 2L of LB media. The culture was grown at 37 °C, 270 rpm until the O.D 600= 0.6-0.8. Cells were collected via centrifuge (4000 RCF, 20 minutes, 4 °C) and weighted. If the combined mass of the cell pellets was below 10 grams, more culture was grown until the cell weighted more than 10 grams. After harvesting, cell pellets were then stored at -80 °C until the next day. Frozen cell pellets were lightly thawed on ice and resuspended in 50 mL of suspension buffer and transferred to a Nalgene Oak Ridge centrifuge tube. Cell was then lysed by sonication (10s ON / 10s OFF, 40% amplitude, 4 minutes) on ice with additions of splashes of brine. Cell debris was then removed via centrifugation (20,000 RCF, 20 minutes, 4°C). The collected supernatant was fractionated by the addition of an equivalent amount of high salt buffer. Lysate was clarified one more time via centrifugation (20,000 RCF, 20 minutes, 4 °C) and filtered through a 0.45 pm PTFE filter.

[0118] Subsequently, ribosome was purified using hydrophobic chromatography (3x5 mL HiTrap Butyl HP column (VWR)) with Fast Performance Liquid Chromatography (FPLC) (Akta Purifier) at 4 °C and with a flow rate of 4 mL per minute. Prepared lysate was loaded onto the column after equilibrating with 60 mL of buffer C. Purification followed with washing with 45 mL of buffer 1 (100% buffer C) followed by 75 mL of wash buffer 2 (80% buffer C, 20% buffer D). Elution of ribosome with 60 mL of ribosome elution buffer (50% buffer C, 50% buffer D), followed by 60 mL of final elution buffer (100% buffer D). Fractions of ribosome with strong absorbance at 280 nm were pooled together. The column recovered by washing with 1 M NaOH and 0.1 M acetic acid, and then stored in 20% ethanol.

[0119] With ultra-centrifugal polycarbonate tubes of 32 mL void volume, 15 mL of cushion buffer was added first. The recovered fraction of ribosome was then overlaid on top of the cushion buffer with an equivalent amount of cushion buffer. The ribosome was pelleted by ultracentrifugation (100,000 RCF, 16h, 4 °C). The pellets were then washed two times with 500 pL of ribosome buffer and resuspended with a magnetic stirrer with 200 pL of ribosome buffer. The ribosome solution was concentrated using an Amicon Ultra filter unit with 3000 MWCO by centrifugation (14,000 RCF, 10 minutes, at 4 °C). The concentration of ribosome was measured using absorbance at 260 nm. For an absorbance of 10 with 1 : 100 dilution, the concentration corresponds to 23 pM. The final ribosome working solution was diluted to 10 pM and stored at -80 °C for further usage. Bradford assay for protein quantification: Bovine gamma globulin was used as a standard. A calibration curve ranging from 0 to 20 pg / mL was constructed. For measurement, the purified protein mixture was diluted 1 : 15, and 5 pL of the diluted sample was added to 250 pL of Bradford reagent in a 96-well microassay plate. The plate was then incubated at room temperature with light shaking to avoid bubbles. Absorbance at 595 nm was then measured using a plate reader.

[0120] Cell-free protein synthesis and characterization: For 10 pL reaction scale without unnatural amino acid incorporation, 2.5 pL of 4x Energy mix (Table 4) was mixed with 1.8 pL of 10 pM Ribosome and 1.3 pL of One Pot protein factors. DNA plasmid template was added with the final concentration of 50 ng / pL. For unnatural amino acid incorporation, purified orthogonal tRNA (o-tRNA) and unnatural amino acid were added for a final concentration of 5 M and 0.25 mM, respectively. The mixture was brought to a final volume of 10 pL with the addition of nuclease and protease-free water.

[0121] Short peptide containing C-terminal Flag epitope was purified via Anti -FLAG agarose resin from the CFPS reaction mixture. The purified peptide was then de-salted and subjected to MALD-MS for analysis.

[0122] Quantification of sfGFP expression level was carried out by incubating the CFPS reaction at 37 °C in a plate reader (Infinite 200 Pro, Tecan) for 3h and then measured (excitation and emission: 488 nm and 512 nm for sfGFP). For taking emission (fixed excitation wavelength at 425 nm, and emission scan from 460 to 630 nm) and excitation scan (fixed emission wavelength of 600 nm, and emission scan from 460 to 560 nm) of OFP, the CFPS reaction was incubated 37 °C in a plate reader for 5h and then measured.

[0123] Example 2: Elongation Factor-Proline Experiment

[0124] L-Proline is the only cyclic amino acid among the 20 proteogenic amino acids. During translation elongation process the ribosome can be stalled by proline’s unfavorable geometry, especially during runs of prolines in proteins. To overcome this challenge, nature utilizes an additional translation factor, elongation factor-proline (EF-P), to reduce stalling. The EF-P factor requires post-translational modification by three proteins: EpmA (YjeA), EpmB (YjeK), and EpmC (YfcM) to yield mature and functional EF-P. To determine if fundamental aspects of translation could be further modified in the One Pot PURE system, EF-P was introduced as a factor for the translation of proteins with multiple, consecutive proline residues. Single plasmid construct of histidine-tagged EF-P, EpmA, EpmB, and EpmC was generated and introduced into a One Pot PURE preparation (Figure 12a). EF-P concentrations were titrated ranging from 500%, 750%, 100%, and 1250% comparing to standard volume overnight culture added into one Pot mixed co-culture. Additionally, we used a sfGFP fusion with truncated basic salivary proline-rich protein 4 at the N-terminal to assess the activity of EF-P in One Pot cell-free protein synthesis reaction (Figure 12b). No statistically difference was observed in the sfGFP fusion production comparing to One Pot cell-free reaction with no EF-P translation factor (Figure 12c, 12d). Thus, the investigation in the addition of EF-P into the One Pot PURE system was not successful. This indicates that not all translation factors can be introduced into the One Pot PURE system without optimization.

[0125] TABLES

[0126] Table 1: Volume of each cells expressing various transcriptional and translational enzymes to be added into the One Pot co-culture for 250 mL scale. All volume indicated above are in pL units.

[0127] Table 2: Buffer recipe for One Pot protein factor purification

[0128] Table 3: Buffer recipe for ribosome purification

[0129] Table 4: 4x Energy mixture recipe Table 5: Primers sequence to generate DNA template for tRNA and short peptide containing C-terminal FLAG epitope

[0130] Reporter sequences:

[0131] Flag-peptide: X=amber or opal or ochre codon. MKKKXDYDYKDDDDK (SEQ ID NO: 19)

[0132] Super folded Green Fluorescent Protein (sfGFP) (SEQ ID NO: 20):

[0133] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGK

[0134] LPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYK

[0135] TRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA

[0136] NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRD

[0137] HMVLHEYVNAAGITWSHPQFEKDYKDDDDK.

[0138] Super folded Green Fluorescent Protein containing in-frame ochre stop codon (X) (sfGFP) (SEQ ID NO: 21):

[0139] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGK

[0140] LPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYK

[0141] TRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHXVYITADKQKNGIKA

[0142] NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRD

[0143] HMVLHEYVNAAGITWSHPQFEKDYKDDDDK.

[0144] Orange fluorescent protein (OFP): X= amber codon (SEQ ID NO: 22)

[0145] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG

[0146] KLPVPWPTLVTTLAXGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFEDDGYY

[0147] KTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

[0148] VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQCALSKDPNEKRDHM

[0149] VLLEFVTAAGITHDMDELYKGSGHHHHHH

[0150] EXEMPLARY ASPECTS

[0151] In view of the described processes and compositions, herein below are described certain more particularly described aspects of the disclosures. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0152] Example 1. A method of producing a polypeptide comprising at least one non- naturally occurring amino acid, wherein the polypeptide is produced using One Pot PURE (Protein Synthesis Using Recombinant Elements) system.

[0153] Example 2. The method of any one of the examples herein, particularly example 1, wherein the One Pot PURE system comprises coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask.

[0154] Example 3. The method of any one of the examples herein, particularly example 2, wherein at least one of the protein-producing cells has been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis.

[0155] Example 4. The method of any one of the examples herein, particularly example 3, wherein the cells are E. coli.

[0156] Example 5. The method of any one of the examples herein, particularly example 3 or 4, wherein between 30 and 40 E. coli clones are used.

[0157] Example 6. The method of any one of the examples herein, particularly example 5, wherein about 36 clones are used.

[0158] Example 7. The method of any one of the examples herein, particularly examples 2- 6, wherein, after coculturing and inducing all protein-producing cells, proteins produced by the cells are purified.

[0159] Example 8. The method of any one of the examples herein, particularly example 7, wherein proteins are purified using single nickel nitrilotriacetic acid (Ni-NTA) purification.

[0160] Example 9. The method of any one of the examples herein, particularly examples 1- 8, wherein the non-naturally occurring amino acid is incorporated by using an orthogonal tRNA synthetase.

[0161] Example 10. The method of any one of the examples herein, particularly example 9, wherein the non-naturally occurring amino acid is levodopa (L-DOPA) or N-e- propagy 1 oxy carb ony 1 -L-ly sine (ProgK) .

[0162] Example 11. The method of any one of the examples herein, particularly examples 1- 10, wherein two or more non-natural amino acids are incorporated. Example 12. The method of any one of the examples herein, particularly examples 9- 11, wherein codon suppression is used in incorporation of the non-naturally occurring amino acid.

[0163] Example 13. The method of any one of the examples herein, particularly examples 9- 11, wherein a stop codon is suppressed.

[0164] Example 14. The method of any one of the examples herein, particularly examples 3-

[0165] 13, wherein at least one expression plasmid has been engineered so that all genes of the plasmid are under the control of the T7 RNA polymerase promoter and an adjacent TetR operator are expressed.

[0166] Example 15. The method of any one of the examples herein, particularly examples 3-

[0167] 14, wherein a Tet repressor is included on at least one expression plasmid.

[0168] Example 16. The method of any one of the examples herein, particularly examples 3-

[0169] 15, wherein plasmids encoding translation factors are transformed into an E. coli BL21 Marionette strain, which contains a genome-encoded array of 12 different, constitutively expressed repressors including Lacl.

[0170] Example 17. A One Pot PURE system configured to produce a non-naturally occurring amino acid.

[0171] Example 18. The system of any one of the examples herein, particularly example 17, wherein the system comprises cells which have been modified with expression plasmids to produce proteins needed for protein synthesis.

[0172] Example 19. The system of any one of the examples herein, particularly example 18, wherein the cells are E. coli.

[0173] Example 20. The system of any one of the examples herein, particularly example 18 or 19, wherein between 30 and 40 E. coli clones are used.

[0174] Example 21. The system of any one of the examples herein, particularly example 20, wherein about 36 clones are used.

[0175] Example 22. The system of v examples 17-21, wherein, after coculturing and inducing all protein-producing cells, proteins produced by the cells are purified.

[0176] Example 23. The system of any one of the examples herein, particularly example 22, wherein proteins are purified using single nickel nitrilotriacetic acid (Ni-NTA) purification.

[0177] Example 24. The system of any one of the examples herein, particularly examples 18- 24, wherein the non-naturally occurring amino acid is incorporated by using an orthogonal tRNA synthetase. Example 25. The system of any one of the examples herein, particularly example 24, wherein codon suppression is used in incorporation of the non-naturally occurring amino acid.

[0178] Example 26. The system of any one of the examples herein, particularly examples 18-

[0179] 25, wherein at least one expression plasmid has been engineered so that all genes of the plasmid are under the control of the T7 RNA polymerase promoter and an adjacent TetR operator are expressed.

[0180] Example 27. The system of any one of the examples herein, particularly examples 18-

[0181] 26, wherein a Tet repressor is included on at least one expression plasmid.

[0182] Example 28. The system of any one of the examples herein, particularly examples 18-

[0183] 27, wherein plasmids encoding translation factors are transformed into an E. coli BL21 Marionette strain, which contains a genome-encoded array of 12 different, constitutively expressed repressors including Lacl.

[0184] Example 29. A method of producing a polypeptide comprising at least one non- naturally occurring amino acid, wherein the method comprises: (a) coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask, wherein at least one of the protein-producing cells have been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis; (b) purifying the proteins produced in step a); (c) introducing to the proteins of step b) to components needed for introduction of a non-naturally occurring amino acid, thereby producing a polypeptide with a non-naturally occurring amino acid.

[0185] Example 30. The method of any one of the examples herein, particularly example 1, wherein production of at least one non-naturally occurring amino acid is optimized.

[0186] Example 31. The method of any one of the examples herein, particularly example 30, wherein the method is optimized by introducing one or more clones in different ratios during co-culturing and comparing results to determine which has greater yield of non-naturally occurring amino acid.

[0187] Example 32. The method of any one of the examples herein, particularly example 31, wherein yield comprises more insertions of non-naturally occurring amino acid in polypeptide, or more overall polypeptide with non-naturally occurring amino acid incorporated. REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A method of producing a polypeptide comprising at least one non-naturally occurring amino acid, wherein the polypeptide is produced using One Pot PURE (Protein Synthesis Using Recombinant Elements) system.

2. The method of claim 1, wherein the One Pot PURE system comprises coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask.

3. The method of claim 2, wherein at least one of the protein-producing cells has been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis.

4. The method of claim 3, wherein the cells are E. coli.

5. The method of claim 3 or 4, wherein between 30 and 40 E. coli clones are used.

6. The method of claim 5, wherein about 36 clones are used.

7. The method of any one of claims 2-6, wherein, after coculturing and inducing all protein-producing cells, proteins produced by the cells are purified.

8. The method of claim 7, wherein proteins are purified using single nickel nitrilotriacetic acid (Ni-NTA) purification.

9. The method of any of claims 1-8, wherein the non-naturally occurring amino acid is incorporated by using an orthogonal tRNA synthetase.

10. The method of claim 9, wherein the non-naturally occurring amino acid is levodopa (L- DOPA) or N-e-propagyloxycarbonyl-L-lysine (ProgK).

11. The method of any one of claims 1-10, wherein two or more non-natural amino acids are incorporated.

12. The method of any one of claims 9-11, wherein codon suppression is used in incorporation of the non-naturally occurring amino acid.

13. The method of any one of claims 9-11, wherein a stop codon is suppressed.

14. The method of any one of claims 3-13, wherein at least one expression plasmid has been engineered so that all genes of the plasmid are under the control of the T7 RNA polymerase promoter and an adjacent TetR operator are expressed.

15. The method of any one of claims 3-14, wherein a Tet repressor is included on at least one expression plasmid.

16. The method of any one of claims 3-15, wherein plasmids encoding translation factors are transformed into an E. coli BL21 Marionette strain, which contains a genome- encoded array of 12 different, constitutively expressed repressors including Lacl.

17. A One Pot PURE system configured to produce a non-naturally occurring amino acid.

18. The system of claim 17, wherein the system comprises cells which have been modified with expression plasmids to produce proteins needed for protein synthesis.

19. The system of claim 18, wherein the cells are A. coli.

20. The system of claim 18 or 19, wherein between 30 and 40 E. coli clones are used.

21. The system of claim 20, wherein about 36 clones are used.

22. The system of any one of claims 17-21, wherein, after coculturing and inducing all protein-producing cells, proteins produced by the cells are purified.

23. The system of claim 22, wherein proteins are purified using single nickel nitrilotriacetic acid (Ni-NTA) purification.

24. The system of any of claims 18-23, wherein the non-naturally occurring amino acid is incorporated by using an orthogonal tRNA synthetase.

25. The system of claim 24, wherein codon suppression is used in incorporation of the non- naturally occurring amino acid.

26. The system of any one of claims 18-25, wherein at least one expression plasmid has been engineered so that all genes of the plasmid are under the control of the T7 RNA polymerase promoter and an adjacent TetR operator are expressed.

27. The system of any one of claims 18-26, wherein a Tet repressor is included on at least one expression plasmid.

28. The system of any one of claims 18-27, wherein plasmids encoding translation factors are transformed into an E. coli BL21 Marionette strain, which contains a genome- encoded array of 12 different, constitutively expressed repressors including Lacl.

29. A method of producing a polypeptide comprising at least one non-naturally occurring amino acid, wherein the method comprises:(a) coculturing and inducing all protein-producing cells needed for protein synthesis in a single flask, wherein at least one of the protein-producing cells have been modified with an expression plasmid, wherein said expression plasmid has been engineered to produce proteins needed for protein synthesis;(b) purifying the proteins produced in step a);(c) introducing to the proteins of step b) to components needed for introduction of a non-naturally occurring amino acid, thereby producing a polypeptide with a non- naturally occurring amino acid.

30. The method of claim 1, wherein production of at least one non-naturally occurring amino acid is optimized.

31. The method of claim 30, wherein the method is optimized by introducing one or more clones in different ratios during co-culturing and comparing results to determine which has greater yield of non-naturally occurring amino acid.

32. The method of claim 31, wherein yield comprises more insertions of non-naturally occurring amino acid in polypeptide, or more overall polypeptide with non-naturally occurring amino acid incorporated.

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