Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (α1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins. Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (α1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins. prolyl-tRNA synthetase trans-4-hydroxyproline ampicillin transforming growth factor glutathioneS-transferase high pressure liquid chromatography isopropyl-1-thio-β-d-galactopyranoside. Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies. Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitroacylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in site-specific incorporation (1Mendel D. Cornish V.W. Schultz P.G. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 435-462Crossref PubMed Scopus (153) Google Scholar) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency.In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2Liu D.R. Magliery T.J. Pastrnak M. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10092-10097Crossref PubMed Scopus (164) Google Scholar, 3Köhrer C. Xie L. Kellerer S. Varshney U. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14310-14315Crossref PubMed Scopus (58) Google Scholar, 4Liu D.R. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4780-4785Crossref PubMed Scopus (152) Google Scholar), but experimental hurdles inherent to this approach have not yet been fully overcome. In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5Yoshikawa E. Fournier M.J. Mason T.L. Tirrell D.A. Macromolecules. 1994; 27: 5471-5475Crossref Scopus (89) Google Scholar) and tryptophan (6Hogue C.W.V. Rasquinha I. Szabo A.B. MacManus J.P. FEBS Lett. 1992; 310: 269-272Crossref PubMed Scopus (78) Google Scholar). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type synthetase. The second requirement, although it is a prerequisite for analogue incorporation, is only beginning to be addressed (4Liu D.R. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4780-4785Crossref PubMed Scopus (152) Google Scholar). Escherichia coli prolyl-tRNA synthetase (ProRS)1 is particularly intriguing with respect to these considerations. ProRS will activate several proline analogues, and some of these can be incorporated into heterologously produced proteins in E. coli. These analogues include l-azetidine-2-carboxylic acid (7Deming T.J. Fournier M.J. Mason T.L. Tirrell D.A. Macromolecules. 1996; 29: 1442-1444Crossref Scopus (42) Google Scholar, 8Fowden L. Richmond M.H. Biochim. Biophys. Acta. 1963; 71: 459-461Crossref Scopus (111) Google Scholar) and 3,4-dehydroproline (9Fowden L. Neale S. Tristram H. Nature. 1963; 199: 35-38Crossref PubMed Scopus (16) Google Scholar, 10Deming T.J. Fournier M.J. Mason T.L. Tirrell D.A. Polym. Mater. Sci. Eng. Proc. 1994; 71: 673-674Google Scholar). In general, proline analogues demonstrated to be incorporated in vivo into proteins are activatedin vitro by ProRS at a rate approaching that of proline (11Papas T.S. Mehler A.H. J. Biol. Chem. 1970; 245: 1588-1595Abstract Full Text PDF PubMed Google Scholar). In contrast to the proline analogues listed above,trans-4-hydroxyproline (Hyp) has been reported not to be activated by ProRS (11Papas T.S. Mehler A.H. J. Biol. Chem. 1970; 245: 1588-1595Abstract Full Text PDF PubMed Google Scholar), and incorporation of Hyp into E. coli-produced proteins has not been demonstrated (10Deming T.J. Fournier M.J. Mason T.L. Tirrell D.A. Polym. Mater. Sci. Eng. Proc. 1994; 71: 673-674Google Scholar). In none of these cases has the intracellular concentration of the analogue necessary to support expression and incorporation into protein been determined. Here we demonstrate that wild-type ProRS can be exploited to incorporate co-translationally trans-4-hydroxyproline efficiently in vivo into recombinant proteins. We reasoned that even a minimal rate of misactivation and misacylation of Hyp by ProRS would be sufficient for incorporation if high intracellular concentrations of Hyp could be achieved. We focused our attention, therefore, on manipulation of E. coli proline transport systems to effect the intracellular accumulation of Hyp. We show that a simple change in E. coli culture conditions results in the intracellular accumulation of Hyp to levels that support synthesis of Hyp-containing recombinant proteins. The initial goal was to produce, in E. coli, Hyp-containing human Type I collagen and collagen-like proteins. In addition to the collagens, our results should make it possible to insert Hyp into any protein synthesized inE. coli and furthermore may have applicability to other amino acid analogues. The gene for prolyl-tRNA synthetase was cloned fromE. coli strain XL-1 Blue (Stratagene, La Jolla, CA) by polymerase chain reaction with primers designed from the published gene sequence (GenBanktm accession no. X55518). The 5′ primer added a flanking NcoI recognition site and the 3′ primer a flanking HindIII recognition site. The PCR product was digested with both NcoI and HindIII and ligated into NcoI/HindIII-digested pTrc99+ expression vector (Amersham Biosciences). After expression, ProRS was purified to homogeneity according to published procedures (12Liu H. Musier-Forsyth K. Biochemistry. 1994; 33: 12708-12714Crossref PubMed Scopus (46) Google Scholar). The gene for the Type I α1 collagen polypeptide was cloned by polymerase chain reaction of the gene from mRNA isolated from human foreskin cells (HS27/ATCC 1634) with primers designed from the published gene sequence (GenBanktm accession no. Z74615). The 5′ primer added a flanking EcoRI recognition site and the 3′ primer a flanking HindIII recognition site. The gene was cloned into theEcoRI/HindIII site of plasmid pBSKS+ (Stratagene), four mutations corrected using the ExSite kit (Stratagene, La Jolla, CA), the sequence confirmed by dideoxy sequencing, and finally the EcoRI/XhoI fragment was subcloned into plasmid pGEX-4T.1 (Amersham Biosciences). The genes for the collagen-binding domain of human fibronectin (13Kornblihtt A.R. Umezawa K. Vibe-Pedersen K. Baralle F.E. EMBO J. 1985; 4: 1755-1759Crossref PubMed Scopus (468) Google Scholar) and the mature transforming growth factor β1 (TGF-β1) (14Derynck R. Jarrett J.A. Chen E.Y. Eaton D.H. Bell J.R. Assoian R.K. Roberts A.B. Sporn M.B. Goeddel D.V. Nature. 1985; 316: 701-705Crossref PubMed Scopus (1383) Google Scholar) and mature bone morphogeneic protein (15Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3365) Google Scholar) polypeptides were expressed from vector pTrcHis (Invitrogen). The plasmids were a generous gift from Dr. Jane Brokaw. Mannose-binding protein was expressed from plasmid pMAL-c2 (New England Biolabs, Beverly, MA). GST was expressed from plasmid pGEX-4T.1. ATP-PPi exchange assays were performed at 2 mm ATP as described (16Heacock D. Forsyth C.J. Shiba K. Musier-Forsyth K. Bioorg. Chem. 1996; 24: 273-289Crossref Scopus (108) Google Scholar). The second order rate constant (k cat/K m) for activation of Hyp by E. coli ProRS was calculated from the slopes of plots of the initial activation rates at Hyp concentrations between 5 and 50 mm and an enzyme concentration of 5 nm. The reported value is an average of three determinations. Because radioactively labeled hydroxyproline of sufficient specific activity is not available, we could not determine the kinetics of the aminoacylation of tRNAPro with Hyp. E. colistrain JM109 was cured of its proline auxotrophy-complementing episome by treatment with acridine orange (17Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). Loss of the episome was confirmed by the inability of JM109 (F−) to grow on minimal medium that lacked proline. In expression experiments, cultures of JM109 (F−) that harbored the expression plasmid in Luria broth (LB) media containing 100 μg/ml ampicillin (Amp) were grown overnight. Cultures were centrifuged and the cell pellets washed twice with M9 minimal media that contained 100 μg/ml Amp and supplemented with 0.5% glucose and 100 μg/ml of all amino acids except glycine and alanine, which were at μg/ml and contained no proline. The cells were finally in the After at for or was added when After of the cultures were by A culture of JM109 (F−) that harbored plasmid in and contained 100 μg/ml Amp was used to 20 cultures of to A of The cultures were grown with at between and were by and washed with of M9 cell was in 20 of media supplemented with 0.5% glucose and 100 μg/ml of all of the amino acids except proline. Cultures were grown at for to proline. After was added to the Hyp was added to mm and to After at cells from three of culture were on and washed twice with 5 of M9 media that contained 0.5% glucose and the appropriate concentration of were with of by for at were to in 100 of and for Hyp by the of and J. Biol. Chem. Full Text PDF PubMed Google Scholar). protein was with the kit cell by three The are the means of three of protein was in in a with After the addition of a of the was and with was to for at which the was in The was in acid and purified on a 5 as a at acid a of acid a with in acid in of at the of the Hyp residues. of proteins has been Chem. PubMed Scopus Google Scholar). Amino acid was at the M. (New on a exchange with was performed at this on an with an were with a by were on an Molecular and A cell was After a at were from to using and the was in from to with a between were at nm. The was calculated using the by We reasoned that requirements were necessary to the promiscuity of E. coli ProRS to tRNAPro that of Hyp at proline codons would occur The first requirement was the of ProRS to activate Hyp and tRNAPro with Hyp. We the activation of Hyp by ProRS and that hydroxyproline is a substrate for activation by E. for Hyp is to be at was not to for activation at hydroxyproline concentrations sufficiently this concentration for a more of the The specificity (k cat/K m) for activation of hydroxyproline and proline by ProRS are and (11Papas T.S. Mehler A.H. J. Biol. Chem. 1970; 245: 1588-1595Abstract Full Text PDF PubMed Google Scholar), The of these is a of the of hydroxyproline as a substrate with proline A. and H. and Scholar). Hyp is activated in vitro by E. coli ProRS of efficiently than proline. 100 mm amino acid the rate of activation of by ProRS is with that of Hyp The of E. coli ProRS for Hyp and in the activation is with that by other E. coli aminoacyl-tRNA that activate amino acids H. E. Rev. 1992; PubMed Google Scholar). Because of experimental we were not to directly the transfer of activated Hyp to In several cases of the activated or amino acid into a protein. E. coli ProRS can tRNAPro Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) but not I. C. A. A. H. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (58) Google Scholar). has not been demonstrated for and because E. coli ProRS is generally and Hyp is not present in E. coli, we on the that E. coli ProRS would aminoacylate tRNAPro with Hyp and would not either Hyp or A second requirement must be met by in vivo approaches to amino acid analogue intracellular concentrations of the analogue sufficient to misacylation of the tRNA must be achieved. Because Hyp is activated in vitro by ProRS with we that high in of hydroxyproline would be necessary to in enough tRNAPro to support protein this we of the phenomenon that proline and other are in to hyperosmotic in E. coli and other J.M. J. Biol. 1988; PubMed Scopus Google Scholar, E. J. 1996; PubMed Google Scholar). by the and genes and are in to J.M. J. Biol. 1988; PubMed Scopus Google Scholar, J. J. 1985; PubMed Google Scholar, J. J. PubMed Google Scholar). accumulation of Hyp coli has not been although Hyp proline by E. coli under conditions I. Tristram H. J. PubMed Google Scholar). These to that E. coli would accumulate Hyp in of proline if in hyperosmotic in media that proline but contain mm coli proline strain JM109 (F−) Hyp when in concentrations of The intracellular hydroxyproline concentration is to the concentration as high as concentrations than because of of the proline a typical value for the of an E. the intracellular concentration of hydroxyproline at mm is This concentration an the concentration of mm and is with the for proline under conditions J.M. J. Google Scholar). this concentration approaches the for the of hydroxyproline activation by The the intracellular hydroxyproline in JM109 (F−) in an adequate of hydroxyproline tRNAPro to support synthesis of a recombinant protein. this we expression of a fragment of the human Type I α1 collagen chain to the of GST in JM109 (F−) under conditions of hyperosmotic The collagen fragment the amino acids of the triple and the acid the that expression of the gene would be limited by in inE. coli with we synthesized the gene from designed to E. coli 1992; PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar). is efficiently expressed in JM109 (F−) in minimal media proline but supplemented with Hyp and a concentration of expression is minimal at Hyp concentrations than the expression at Hyp concentrations than 20 at a Hyp concentration of concentrations than mm in protein and expression at concentrations than mm The sodium chloride concentrations that for the accumulation of to that the intracellular concentration of hydroxyproline to which that expression is limited in this by the intracellular accumulation of or can be for as the the intracellular accumulation of Hyp is the of expression than the of the the of in GST and in in its and growth it is expressed at of the protein. proteins of than that were of or or mRNA were not of Hyp-containing in JM109 of expression of on The concentration of hydroxyproline is was added to mm in and was with mm the of expression of in hyperosmotic 2 and and added The and quantities of are of the other was added at and all cultures except that in were with mm The the of In the in we that Hyp would be at of the proline codons of protein was with at GST and at the at the of and was purified by of this protein a to a of This is of that for if it Hyp in of proline. was not in amino acid of purified a with substitution of Hyp for proline. that Hyp substitution only at proline we the amino acids of The first codons of the protein sequence The sequence was these results that Hyp was only at proline codons and that the of the E. machinery was not altered by either the high intracellular concentration of Hyp or hyperosmotic culture of our was to the methodology to produce Hyp-containing collagen polypeptides in E. coli. The of the is the In collagens, Hyp in the is for the of a triple structure and Biophys. PubMed Scopus Google is not in in collagens, but is in this in from The which Hyp only in not triple under conditions in which K. S. Biophys. PubMed Scopus Google Scholar). the of Hyp on triple is and the a triple J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Our methodology not between proline codons and will insert Hyp at any proline in either or these we were in the of Hyp in both the and as in In a of a triple and the of Google Scholar). A at and a at this was to for 5 the was the of the at was and the at was This is typical of the triple structure of collagen and the of Google Scholar). A of in a of A fragment of the of the Type I α1 collagen chain in to forms with a of A. E. R. Biochemistry. 1996; PubMed Scopus Google Scholar). global Hyp for proline substitution in both the forms triple of to of collagen containing Hyp at the and only in the These results support the that Hyp in the not the triple a but in specific to triple Because forms a triple but Pro in the may the effect of Hyp in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that Hyp in with respect to amino however, because of global Hyp for Pro substitution no triplets occur with Hyp and This with the in Hyp in may to its triple The human Type I α1 and collagen polypeptides and although more than four the of as with GST in JM109 (F−) in media 5 the genes for the collagen of these proteins were from designed to in coli In expression from a Type I α1 gene to in coded amino acid sequence but containing the human could not be in of cell of JM109 cultures 5 sequence or between the genes for and are of expression in E. coli. This is the of in these genes and of in tRNA levels in E. coli with on other or be in either hyperosmotic media containing only proline or in media not and are when expressed in Hyp-containing media at either or and may from the Hyp-containing proteins may because a the proteins The of codons for E. in the human gene and the of collagen polypeptides in E. coli in expression of human collagen in E. coli has not been reported We to our by the expression of other proteins in media 5 In addition to the mature a fragment of human a of fibronectin and the mature and a of fibronectin and the mature bone morphogeneic protein 2 polypeptide are expressed in JM109 (F−) in In expression is on and Hyp. The of in these proteins between (TGF-β1) and These with expression of containing that any of can be with the protein inE. coli. not on the post-translational enzymatic of proline to and as for a at a translational than post-translational substitution not on the or activity of the recombinant protein. In media that contain a of proline and the of which of these amino acids is at a proline should on the of aminoacylation of tRNAPro with the amino of the appropriate of proline to hydroxyproline in the growth media should make it possible to proteins that contain any of determine this is the we expressed GST in JM109 (F−) in media containing a of hydroxyproline but amounts of proline In medium containing either no proline or the of GST are that these proteins contain amounts of mm GST at the as GST expressed in media containing proline but no Hyp. proline concentrations in the of to the of the protein is between these which that these proteins contain amounts of Hyp and proline. We that substitution of hydroxyproline for proline at or proline in media containing mm Hyp. The of proline to hydroxyproline in the media at this concentration is This is to the of vitro specificity for activation of these amino acids by ProRS which in the of the incorporation the in vivo kinetics of activation of the of hydroxyproline with genetic approaches to insert site-specific Hyp at of the may make it possible to produce human with any Hyp This is an simple and to insert Hyp into any protein that can be synthesized in E. coli. The of the results from the use of DNA coding triplets and the wild-type amino acid In the general it is that only misacylation of a tRNA with an analogue is a sufficient intracellular concentration of the amino acid analogue can be achieved. of amino acid transport systems to intracellular accumulation of the analogue may be of general use and may be with amino acid analogues other than Hyp. A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), alter in to and conditions may be in these systems to the and incorporation of novel amino acid analogues. can protein and of proline can be the in protein K. H. H. J. 1994; PubMed Scopus Google Scholar, K. J. Biol. PubMed Scopus (16) Google Scholar). J. J. Chem. 1996; PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar) that of on the proline the rate and triple These are not to be specific to collagen and that hydroxyproline may or the rate of protein in proteins. The to produce quantities of Hyp-containing proteins inE. coli should make possible biophysical and biochemical approaches to these and may the to novel proteins with or We R. S. of K. Musier-Forsyth of J. D. and for and on the We R. for with the