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Dual charging system for selectively introducing non-native amino acids into proteins using an in vitro synthesis method


Title: Dual charging system for selectively introducing non-native amino acids into proteins using an in vitro synthesis method.
Abstract: This invention provides for a novel means of incorporating non-native amino acids into preselected positions of a protein using a cell-free synthesis system. The methods involve the use of non-orthogonal, native isoaccepting sense tRNAs that are encoded by the genetic code. Such methods allow for numerous non-native amino acids to be incorporated through the use of sense codons without having to rely upon orthogonal tRNA-synthetase pairs. ...

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USPTO Applicaton #: #20100184134 - Class: $ApplicationNatlClass (USPTO) -
Inventors: Alexei M. Voloshin, James F. Zawada, Daniel Gold, Christopher James Murray, James Edward Rozzelle, Nathan Uter, Gang Yin



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The Patent Description & Claims data below is from USPTO Patent Application 20100184134, Dual charging system for selectively introducing non-native amino acids into proteins using an in vitro synthesis method.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §1.119(e) of U.S. Application Nos. 61/144,083, 61/144,097 and 61/144,030, all filed Jan. 12, 2009, each of which is incorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK.

NOT APPLICABLE

BACKGROUND OF THE INVENTION

- Top of Page


Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, vaccines, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using lysates derived from cells.

Because only twenty amino acids are naturally incorporated into proteins, limitations to the production of a desired protein exist. For example, a peptide that is potentially useful as a therapeutic agent may be quickly degraded or otherwise inactivated upon administration to a patient as a result of proteases present within the patient. Likewise, infectious agents such as bacteria or viruses are more likely to develop resistance against peptides that contain only naturally occurring amino acids. This occurs because enzymes that are produced by the bacteria or virus that can inactivate a peptide drug are more likely to inactivate a peptide containing naturally occurring amino acids as opposed to a peptide containing non-native amino acids. Such limitations become even more apparent when compared with small organic molecule synthesis, in which any structural change can be made to influence functional properties of the compound. As a result, proteins containing non-native amino acids are becoming more auspicious for therapeutic uses. Furthermore, peptides containing non-native amino acids are extremely useful for non-therapeutic research purposes, such as uses relevant to the structural and functional probing of proteins, construction of peptide libraries for combinatorial chemistry, and proteomic studies.

Although the twenty naturally occurring amino acids can be modified by post-translational modification, expanding the genetic code to include additional non-native amino acids with novel biological, chemical, or physical properties will increase the utility of the protein containing such novel non-native amino acids. Protein properties may include the size, acidity, nucleophilicity, hydrogen-bonding, or hydrophobicity of the protein.

Different strategies have been utilized to synthesize peptides containing non-native amino acids. Synthetic peptide chemistry has been used routinely for this purpose. See, e.g., Eckert et al., Cell 99:103-15 (1999). However, routine solid-phase peptide synthesis is generally limited to small peptides with less than 100 residues. With the recent development of enzymatic ligation and native chemical ligation of peptide fragments, it is possible to make larger proteins. However, these methods are not easily scaled. See, e.g., Dawson and Kent, Annu Rev. Biochem. 69:923 (2000).

In vivo translation using living cells is widely used for the efficient synthesis and post-translational modification of proteins from a genetically encoded natural or recombinant DNA sequence. However, folding may be inefficient if the protein is expressed in inclusion bodies. Most importantly, such methods are more difficult for the selective incorporation of multiple non-native amino acids, or to control the post-translational modification process.

In vitro, or cell-free, protein synthesis offers several advantages over conventional in vivo protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous since it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than with in vivo protein synthesis because concerns of cell growth or viability do not exist. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.

The productivity of cell-free systems has improved over 2-orders of magnitude in recent years, from about 5 μg/ml-hr to about 500 μg/ml-hr. Such improvements have made in vitro protein synthesis a practical technique for laboratory-scale research and provides a platform technology for high-throughput protein expression. It further indicates the feasibility for using cell-free technologies as an alternative means to in vivo large-scale, commercial production of protein pharmaceuticals.

The incorporation of non-native amino acids into proteins remains a challenge with both in vivo and in vitro protein synthesis systems. A major hurdle in this field of endeavor is promoting recognition of an aminoacyl-tRNA synthetase with a non-native amino acid. An aminoacyl-tRNA synthetase is an enzyme that catalyzes the bond of a specific amino acid to its cognate tRNA molecule. In most cases, each naturally occurring amino acid has one specific aminoacyl-tRNA synthetase that will aminoacylate that amino acid to its proper tRNA molecule, which is known as tRNA charging. There exists relatively few aminoacyl-tRNA synthetases considering the fact that the degeneracy of the genetic code allows amino acids to be charged to more than one kind of tRNA molecule. Thus, the success of incorporating non-native amino acids into proteins depends on the recognition of the non-native amino acid by aminoacyl-tRNA synthetases, which in general requires high selectively to insure the fidelity of protein translation. The fidelity of aminoacylation is maintained both at the level of substrate discrimination and proofreading of both non-cognate intermediates and protein products.

One strategy has been to incorporate non-native amino acids into proteins using aminoacyl-tRNA synthetases that cannot discriminate between non-native amino acids that are structurally similar to their natural counterparts due to lack of proofreading mechanisms. Because the proofreading activity of the aminoacyl-tRNA synthetase has been disabled, structural analogs of natural amino acids that have been misactivated may escape the editing functions of the synthetase, and be incorporated into the growing peptide chain as desired. See, e.g., Doring et al., Science 292:501 (2001).

A major limitation of the abovementioned strategy is that all sites corresponding to a particular natural amino acid throughout the protein are replaced. The extent of incorporation of the natural and non-native amino acid may also vary because it is difficult to completely deplete the cognate natural amino acid inside the cell. Another limitation is that these strategies make it difficult to study the mutant protein in living cells because the multi-site incorporation of analogs often results in toxicity. Finally, this method is applicable in general only to close structural analogs of the common amino acids, again because substitutions must be tolerated at all sites in the genome.

More recently, orthogonal tRNAs and corresponding orthogonal aminoacyl-tRNA synthetases that charge the orthogonal tRNA with the desired non-native amino acid has been used as a strategy to overcome previous limitations. An orthogonal tRNA is a tRNA that base pairs with a codon that is not normally associated with an amino acid such as a stop codon or 4 base pair codon, etc. Importantly, orthogonal components do not cross-react with any of the endogenous tRNAs, aminoacyl-tRNA synthetases, amino acids, or codons in the host organism.

A commonly used orthogonal system for the incorporation of non-native amino acids is the amber suppressor orthogonal tRNA. Using this system, a suppressor tRNA is prepared that recognizes the stop codon UAG and is chemically aminoacylated with a non-native amino acid. Conventional site-directed mutagenesis is used to introduce the stop codon TAG at the site of interest in the protein gene. When the aminoacylated suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, the non-native amino acid is incorporated in response to the UAG codon which gives a protein containing the non-native amino acid at the specified position. See, e.g., Sayers et al., Nucleic Acids Res. 16:791-802 (1988). Evidence has shown that the desired non-native amino acid is incorporated at the position specified by the UAG codon and that the non-native amino acid is not incorporated at any other site in the protein. See, e.g., Noren et al., Science 244:182-88 (1989); Ellman et al., Science 255: 197-200 (1992). For additional discussion of orthogonal translation systems that incorporate non-native amino acids, and methods for their production and use, see also Wang and Schultz, Chem. Commun. 1:1-11 (2002); Xie and Schultz, Methods 36:227-38 (2005); Xie and Schultz, Curr. Opinion in Chemical Biology 9:548-554 (2005); Wang et al., Annu Rev. Biophys. Biomol. Struct. 35:225-49 (2006); and Xie and Schultz, Nat. Rev. Mol. Cell Biol. 7:775-82 (2006).

However, the incorporation of non-native amino acids using orthogonal components suffers from much lower yields because it relies on inherently inefficient suppressor tRNAs competing with termination factors. In addition, the use of orthogonal components for incorporation of non-native amino acids has been restricted to selective incorporation of only a single non-native amino acid per protein at only one of the three nonsense termination codons (the UAG amber stop codon) because of competition at amino acid sense codons from natural amino acids catalyzed by the tRNA charging and proofreading activities of the twenty different aminoacyl-tRNA synthetases, and because attempts to use a second termination codon (UGA) often fails due to read through by the ribosome. See, e.g., Cload et al., Chem. and Biol. 3:1033-38 (1996).

While some attempts have been made to incorporate non-native amino acids into proteins using tRNAs that recognize sense codons, such attempts have been made using a pure reconstituted in vitro translation system. See Tan et al., Methods 36:279-90 (2005); Forster et al., U.S. Pat. No. 6,977,150. However, such pure reconstituted translation systems require purified translational components, which is impractical outside of the context of research, very expensive, and not shown to be highly efficient.

There exists a need in the art for incorporating non-native amino acids into a growing polypeptide chain, where orthogonal tRNA/aminoacyl-tRNA synthetase pairs can be avoided, where native isoaccepting tRNAs aminoacylated with non-native amino acids recognize sense codons and subsequently incorporate the non-native amino acid into a growing polypeptide chain at a position defined by the sense codon, where numerous non-native amino acids can be incorporated at defined positions, and where a crude cell-free protein synthesis system can be used that avoids the impracticality, expense, and inefficiency of a pure reconstituted in vitro translation system. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.

BRIEF

SUMMARY

- Top of Page


OF THE INVENTION

This invention discloses a method for introducing non-native amino acids into pre-selected positions of a protein using a cell-free synthesis system comprising the steps of 1) obtaining a nucleic acid template comprising degenerate sense codons, 2) lysing a cell population to yield a cell lysate, 3) aminoacylating a first and second isoaccepting sense tRNA in separate tRNA charging reactions, said first isoaccepting sense tRNA charged with an amino acid and said second isoaccepting sense tRNA charged with a non-native amino acid, 4) adding the first and second isoaccepting sense tRNAs charged with their respective amino acids and the nucleic acid template to the cell lysate and permitting the reaction to generate a protein bearing non-native amino acids in positions corresponding to the second sense codons of the template.

More specifically, this invention is an in vitro method of introducing non-native amino acids into pre-selected positions of a protein using a cell-free synthesis system, the method comprising the steps of:

a) Obtaining a nucleic acid template comprising degenerate sense codons where a first sense codon and a second sense codon correspond to a same native amino acid but differ in their respective nucleotide sequence;

b) Generating a cell lysate;

c) Preventing an endogenous native amino acid from incorporating into a growing polypeptide chain at positions corresponding to the first and second sense codons;

d) Adding a first catalytic aminoacylating agent to a first reaction vessel containing a charging reaction mixture including an amino acid and a first isoaccepting sense tRNA said first isoaccepting sense tRNA recognizing the first sense codon;

e) Aminoacylating the first isoaccepting sense tRNA with the amino acid to yield a tRNA:amino acid charged moiety;

f) Adding a second catalytic aminoacylating agent to a second reaction vessel containing a charging reaction mixture including a non-native amino acid and a second isoaccepting sense tRNA said second isoaccepting sense tRNA recognizing the second sense codon;

g) Aminoacylating the second isoaccepting sense tRNA with the non-native amino acid to yield a tRNA:non-native amino acid charged moiety;

h) Combining the cell lysate with: 1) the tRNA:amino acid charged moiety; 2) the tRNA:non-native amino acid charged moiety; and, 3) the nucleic acid template comprising the first and second codons under conditions appropriate to generate a polypeptide from the template; and;

i) Permitting the reaction to generate the polypeptide bearing non-native amino acids in those positions corresponding to the second sense codons of the template.

An endogenous native amino acid can be prevented from being incorporated into the desired polypeptide chain at positions corresponding to the first and second sense codons by depleting the native aminoacyl-tRNA synthetase that aminoacylates the endogenous native amino acid.

The endogenous native amino acid can also be prevented from incorporating into a growing polypeptide chain at positions corresponding to the first and second sense codons by inactivating both a native first isoaccepting sense tRNA that recognizes the first sense codon and a native second isoaccepting sense tRNA that recognizes the second sense codon. This may be accomplished by adding an inactivated aminoacyl-tRNA synthetase that selectively binds to the native first and second isoaccepting sense tRNAs, said inactivated synthetase having the ability to outcompete the native aminoacyl-tRNA synthetase. This may also be accomplished by adding anti-sense DNA that selectively binds to the native first and second isoaccepting sense tRNAs. In some embodiments, the first and second isoaccepting sense tRNAs are inactivated by adding a specific tRNA ribonuclease or active fragments thereof that selectively cleave the native first and second isoaccepting sense tRNAs. In some embodiments, the first and second isoaccepting sense tRNAs are inactivated by adding colicin D or an active fragment of colicin D.

The above-described method can be performed wherein one or both of the catalytic aminoacylating agents are aminoacyl-tRNA synthetases. When the catalytic aminoacylating agents are aminoacyl-tRNA synthetases, said aminoacyl-tRNA synthetases are removed from the reaction vessel of the tRNA charging reaction prior to combining the tRNA:amino acid charged moiety and tRNA:non-native amino acid charged moiety with the cell lysate. The catalytic aminoacylating agents can also be ribosomes. The above-described method may use a cell population comprises bacterial cells, preferably E. coli cells. The cell population may be depleted for arginine decarboxylase. The cell population comprise rabbit reticulocytes. The above-described method may also utilize a cell lysate that exhibits active oxidation phosphorylation during protein synthesis.

In a related method of this invention, the cell lysate is depleted of the native aminoacyl-tRNA synthetase by genetically altering the cells prior to lysing where the alteration replaces the gene encoding the native aminoacyl-tRNA synthetase with a gene encoding an aminoacyl-tRNA synthetase fused to a capture moiety. The native aminoacyl-tRNA synthetase tagged with a capture moiety may be heterologous to the host cell population. The above-described method may comprise the step of capturing the native aminoacyl-tRNA synthetase fused to a capture moiety by affinity chromatography. The affinity chromatography method may be immunoaffinity chromatography. The capturing of the native aminoacyl-tRNA synthetase fused to a capture moiety may occur by immunoprecipitation using an antibody that recognizes the capture moiety.

In a related system, this invention is a cell-free synthesis reaction system for introducing non-native amino acids into preselected positions of a protein comprising: a) a first catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a first isoaccepting sense tRNA with its corresponding amino acid to yield a tRNA:amino acid charged moiety; b) a second catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a second isoaccepting sense tRNA with a non-native amino acid to yield a tRNA:non-native amino acid charged moiety; and c) a reaction vessel containing a cell lysate containing a mixture of reagents able to carry out in vitro synthesis of proteins from a nucleic acid template;

where all three vessels have openings that permit the combining of the two charging mixtures and cell lysate into a single reaction mixture. The above mentioned system may be used with cells derived from a bacterial population, preferably a bacterial population of E. coli cells. The system is optionally practiced using E. coli cells depleted of arginine decarboxylase. The system is further optionally practiced wherein the cell lysate has a functional oxidative phosphorylation system. The system described herein can be used where one or both of the catalytic aminoacylating reagents are either aminoacyl-tRNA synthetase or ribozymes.

The invention further provides a kit for the in vitro synthesis of proteins having non-native amino acids introduced into preselected positions of the protein, the kit comprising: a) a first catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a first isoaccepting sense tRNA with its corresponding amino acid to yield a tRNA:amino acid charged moiety; b) a second catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a second isoaccepting sense tRNA with a non-native amino acid to yield a tRNA:non-native amino acid charged moiety; and c) a reaction vessel containing a cell lysate containing a mixture of reagents able to carry out in vitro synthesis of proteins from a nucleic acid template.

The above mentioned kit may be used with cells derived from a bacterial population, preferably a bacterial population of E. coli cells. The kit is optionally practiced using E. coli cells depleted of arginine decarboxylase. The kit is further optionally practiced wherein the cell lysate has a functional oxidative phosphorylation system. The kit described herein can be used where one or both of the catalystic aminoacylating reagents are either aminoacyl-tRNA synthetase or ribozymes.

BRIEF DESCRIPTION OF THE DRAWINGS

- Top of Page


FIG. 1 shows (a) the tRNAGAAPhe HDV ribozyme plasmid DNA template used for in vitro transcription, (b) a fragment of the DNA template detailing the orientation of the T7 promotor, tRNAGAAPhe coding sequence fused to the hepatitis delta virus (HDV) ribozyme sequence, and (c) the resulting E. coli isoaccepting tRNAGAAPhe transcript secondary structure with the anticodon sequence GAA in red, where the subscript denotes the corresponding anticodon sequence.

FIG. 2 shows the process flow diagram illustrating the steps required to generate novel nnAA-tRNAs including (a) in vitro transcription of tRNA-HDV ribozyme DNA template (b) isolation of tRNA 2,3′-cyclic phosphate by size exclusion chromatography, (c) enzymatic hydrolysis of tRNA 2,3′-cyclic phosphate using T4 polynucleotide kinase (PNK) and, (d) aminoacylation of engineered isoaccepting tRNAs with nnAAs catalyzed by engineered amino acid tRNA synthetase (aaRS) enzymes.

FIG. 3 shows TBE/UREA gels for protocols 1-4 used for optimization of in vitro transcription of two different engineered E. coli tRNACUCGlu constructs. Both constructs are mutated at U34C to produce a CUC anticodon; the rightmost construct contains additional noted mutations that should theoretically increase transcription yield.

FIG. 4 shows TBE/UREA gels of in vitro transcription protocols 1-4 for two different engineered tRNAUUGGln constructs from E. coli and H. pylori, respectively.

FIG. 5 shows (a) an illustration of the Hepatitis Delta Virus (HDV) consensus sequence used for generating 3′ homogeneous tRNA. The autocatalytic ribozyme cleaves at the 3′ end of the tRNA leaving a 2′,3′-cyclic phosphate that is subsequently removed before aminoacylation. (b) Size exclusion chromatographic separation of the transcription product illustrating separation of the 73 nucleotide tRNA 2′,3′-cyclic phosphate product from the self-cleaved HDV ribozyme.

FIG. 6 shows the effect of various additives on RNA stability.

FIG. 7 shows the time dependence of the dephosphorylation of tRNA 2′-3′-cyclic phosphate by PNK treatment as measured by (a) separation of reactant and product using acid/urea gel electrophoresis or (b) using a malachite green phosphate release assay.

FIG. 8 shows IMAC purification profiles of (a) E coli GluRS 6XHis and (b) H pylori GluRS2 (ND) enzymes used for charging tRNA with non-native amino acids (nnAA).

FIG. 9 shows (a) the dimeric structure of the homologous T. thermophilus PheRS illustrating the amino acid recognition site containing A294G and the anti-codon recognition site. (b) IMAC purification profile of cell-free produced PheRS(A294G) showing pull-down of the dimeric complex by the 6X His-tagged PheS(A294G) domain.

FIG. 10 shows the purification of several PheRS(A294G, A794X) variants produced by cell-free protein synthesis.

FIG. 11 shows percent Phe aminoacylation analysis of [30-32Phe-tRNAPhe catalyzed by PheRS(A294G) for 30 min. (a) P1 nuclease digestion of (b) Phe-tRNAAAAPhe, Phe-tRNACUAPhe, or Phe-tRNAGAAPhe results in [32P]Phe-AMP that can be separated from free [32P]AMP on PEI cellulose TLC plates and imaged using autoradiography.

FIG. 12 shows percent para-acetyl Phe (pAF) aminoacylation analysis of pAF-tRNAPhe variants catalyzed by PheRS(A294G) under various conditions as measured by autoradiography using a end-labeled [32P]-3′ tRNA.

FIG. 13 shows the time dependence of the formation of (a) pAF-tRNACUAPhe and (b) pAF-tRNAAAAPhe as measured by autoradiography.

FIG. 14 shows that (a) E. coli GluRS can robustly aminoacylate tRNACUCGlu with cognate glutamate as measured using a [32P]-3′ tRNA end-labeling assay under optimized conditions. Mono-fluoroglutamate (F-Glu) AMP is not separated from [32P]-AMP under these chromatographic conditions. (b) H. pylori GluRS2(ND) can aminoacylate H. pylori tRNACUCGlu with Glu and F-Glu, although F-Glu AMP is not separated from AMP under these chromatographic conditions.

FIG. 15 shows (a) the separation of aminoacylated pAF-tRNAGAAPhe from tRNAGAAPhe using hydrophobic interaction chromatography, and (b) the chromatographic mobility of aminoacylated pAF-tRNAGAAPhe, tRNAGAAPhe, and tRNAGAAPhe2′,3′ cyclic phosphate by acid-urea gel electrophoresis.

FIG. 16 shows (a) the separation of aminoacylated pAF-tRNACUAPhe from tRNACUAPhe and (b) pAF-tRNAAAAPhe from tRNAAAAPhe using hydrophobic interaction chromatography.

FIG. 17 shows that (a) Wild type E. coli tRNAGlu and (b) in vitro transcribed E coli tRNACUCGlu can be robustly charged with mono-fluoroglutamate as determined by acid/urea gel electrophoresis.

FIG. 18 shows cell-free synthesis yields of GMCSF as function of the concentration of added lysine, phenylalanine, or glutamic acid to the extract.

FIG. 19 shows (a) a diagram of the species involved in non-native amino acid incorporation into fluorescent turboGFP in the cell-free synthesis reaction, including background recharging of engineered isoaccepting tRNA by endogenous E coli PheRS, (b) the chemical structure of an active-site directed inhibitor of PheRS, 5′-O-[N-(Phenylalanyl) sulfamoyl] adenosine, Phe-SA, and (c) the determination of IC50 for inhibition of the rate of cell-free synthesis of turboGFP as a function of added inhibitors Phe-SA (IC50=0.8 nM) or Glu-SA (IC50=54 nM).

FIG. 20 shows a response surface describing the relationship between added Phe amino acid and [Phe-SA] inhibitor on the rate of turboGFP cell-free synthesis




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stats Patent Info
Application #
US 20100184134 A1
Publish Date
07/22/2010
Document #
File Date
12/31/1969
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