SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1989287988> ?p ?o ?g. }

Showing items 1 to 100 of ±136 with 100 items per page.

W1989287988 endingPage "17105" @default.
W1989287988 startingPage "17100" @default.
W1989287988 abstract "The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Plückthun, A. (1998) Nature Biotechnol. 16, 376–380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of anskp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containingcis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Plückthun, A. (2000) J. Biol. Chem. 275, 17106–17113), we conclude that the effect of FkpA is independent of its PPIase activity. The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Plückthun, A. (1998) Nature Biotechnol. 16, 376–380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of anskp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containingcis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Plückthun, A. (2000) J. Biol. Chem. 275, 17106–17113), we conclude that the effect of FkpA is independent of its PPIase activity. gene-3 protein single-chain Fv kilobase pair(s) fluorescein isothiocyanate enzyme-linked immunosorbent assay base pair(s) polymerase chain reaction peptidylprolylcis,trans-isomerase FK506-binding protein An important strategy for the production of recombinant disulfide-containing proteins is their secretion to the periplasmic space of Escherichia coli, as the periplasm is the location of the disulfide-forming machinery (1.Bardwell J.C. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (197) Google Scholar, 2.Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar). Antibodies are one class of particularly important disulfide-containing proteins that have frequently been expressed in this compartment, and this is probably the most convenient method to obtain engineered antibodies for research and medical or technological applications (3.Plückthun A. Krebber A. Krebber C. Horn U. Knüpfer R. Wenderoth L. Nieba L. Proba K. Riesanberg D. Antibody Engineering: A Practical Approach. IRL Press Ltd., Oxford1996: 203-252Google Scholar, 4.Huston J.S. McCartney J. Tai M.S. Mottola-Hartshorn C. Jin D. Warren F. Keck P. Oppermann H. Int. Rev. Immunol. 1993; 10: 195-217Crossref PubMed Scopus (150) Google Scholar, 5.Dall'Acqua W. Carter P. Curr. Opin. Struct. Biol. 1998; 8: 443-450Crossref PubMed Scopus (87) Google Scholar). Unfortunately, folding problems of antibodies and other heterologous proteins in the periplasm are frequently observed, and the nature and existence of molecular chaperones in this compartment are still only poorly understood (2.Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar, 6.Wall J.G. Plückthun A. Curr. Opin. Biotechnol. 1995; 6: 507-516Crossref PubMed Scopus (81) Google Scholar, 7.Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (187) Google Scholar).It has been previously shown (8.Skerra A. Plückthun A. Protein Eng. 1991; 4: 971-979Crossref PubMed Scopus (90) Google Scholar, 9.Knappik A. Krebber C. Plückthun A. Bio/Technology. 1993; 11: 77-83Crossref PubMed Scopus (13) Google Scholar, 10.Knappik A. Plückthun A. Protein Eng. 1995; 8: 81-89Crossref PubMed Scopus (218) Google Scholar, 11.Ulrich H.D. Patten P.A. Yang P.L. Romesberg F.E. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11907-11911Crossref PubMed Scopus (84) Google Scholar, 12.Forsberg G. Forsgren M. Jaki M. Norin M. Sterky C. Enhorning A. Larsson K. Ericsson M. Björk P. J. Biol. Chem. 1997; 272: 12430-12436Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) that the functional periplasmic expression of antibody fragments can be limited by the sequence-dependent, moderate efficiency of periplasmic folding, which can furthermore be accompanied by cell lysis, as has also been observed for other proteins (13.Suominen I. Karp M. Lahde M. Kopio A. Glumoff T. Meyer P. Mantsala P. Gene (Amst.). 1987; 61: 165-176Crossref PubMed Scopus (44) Google Scholar). Typically, the actual membrane transport is not limiting (8.Skerra A. Plückthun A. Protein Eng. 1991; 4: 971-979Crossref PubMed Scopus (90) Google Scholar), and large amounts of correctly processed, but precipitated protein are available in the periplasm. It is still unclear which factors, if any, might play a role in preventing the aggregation and in guiding the correct folding of functional antibody fragments or other periplasmic proteins in E. coli, and it is certainly conceivable that different factors play a role for different proteins.We have recently developed a selection system for the identification of novel factors that may help the functional periplasmic expression of a substrate protein in question (14.Bothmann H. Plückthun A. Nature Biotechnol. 1998; 16: 376-380Crossref PubMed Scopus (163) Google Scholar). It was based on the assumption that the folding of soluble periplasmic protein occurs in contact with the same machinery and in the same environment as that of a protein displayed on filamentous phage. Fusion proteins with the minor coat protein gene-3 protein (g3p)1of filamentous phage are transiently produced in a state anchored to the inner membrane, before they are incorporated into the phage coat (15.Davis N.G. Boeke J.D. Model P. J. Mol. Biol. 1985; 181: 111-121Crossref PubMed Scopus (87) Google Scholar). Thus, the folding of the fusion protein occurs in the periplasm and should be influenced by the same factors as that of a soluble periplasmic protein.The selection system works by displaying a poorly expressing antibody fragment on filamentous phage and coexpressing a library of E. coli genes on the same phagemid. Note that these E. coli proteins encoded by the library are not displayed; they are merely expressed by the host cell producing a particular phage in question. If the coexpressed factor improves expression of the antibody-g3p fusion protein, a higher percentage of the phage particles will carry a functional antibody fragment and thus be selectable, even though this displayed antibody fragment is identical in all cases.In a previous study, we identified the periplasmic protein Skp (OmpH, HlpA) by this methodology (14.Bothmann H. Plückthun A. Nature Biotechnol. 1998; 16: 376-380Crossref PubMed Scopus (163) Google Scholar). Since we wanted to select for additional factors that might increase the expression yield, we have repeated this selection in the present study, this time using anE. coli library prepared from a strain devoid of theskp gene. We enriched the gene coding for the protein FkpA, a periplasmic peptidylprolyl cis,trans-isomerase (16.Horne S.M. Young K.D. Arch. Microbiol. 1995; 163: 357-365Crossref PubMed Scopus (84) Google Scholar). Upon characterizing the effect further, we found that only FkpA, but not the other periplasmic peptidylprolyl cis,trans-isomerases PpiA and SurA, had any such effect, suggesting that there is no functional redundancy in these enzymes. Most intriguingly, we found that FkpA has also a very beneficial effect on antibody fragments whose rate-limiting proline isomerization it cannot catalyze and, particularly noteworthy, that do not have any cis-proline at all.DISCUSSIONIn this work, we validated our selection system for periplasmic folding factors. They may be extremely useful in increasing the functional yield of recombinant proteins such as antibodies, which need to be produced in the periplasm because of their disulfide bonds. We believe that this selection approach is very promising for the identification of additional factors involved in periplasmic folding and in preventing misfolding and that this strategy can be expanded to the screening of different genetic libraries for such factors. The protein FkpA, which we have enriched, indeed clearly improves the expression yield of a wide range of functional scFv fragments in the periplasm of E. coli (in some cases, 10-fold). This greatly beneficial effect on the folding yield is visible for all scFv fragments tested, except one, which has good folding properties anyway.FkpA was first described as an E. coli protein homologous to the eukaryotic FK506-binding proteins (FKBPs) (16.Horne S.M. Young K.D. Arch. Microbiol. 1995; 163: 357-365Crossref PubMed Scopus (84) Google Scholar), a class of well characterized PPIases that have been shown to be inhibitable by the macrolide FK506. Mature FkpA is located in the periplasm (21.Missiakas D. Betton J.M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar), and its gene carries ςE-binding sites in the promoter region (40.Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (209) Google Scholar). FkpA shows a high peptidyl cis,trans-isomerase activity, as shown with RNase T1 as substrate (35.Ramm K. Plückthun A. J. Biol. Chem. 2000; 275: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Antibody Folding and Effect of FkpAThe in vitrofolding of antibody scFv fragments containing κ light chains, which normally contain two cis-prolines (positions L8 and L95), has been investigated in detail (34.Ramm K. Gehrig P. Plückthun A. J. Mol. Biol. 1999; 290: 535-546Crossref PubMed Scopus (49) Google Scholar, 36.Jäger M. Plückthun A. J. Mol. Biol. 1999; 285: 2005-2019Crossref PubMed Scopus (42) Google Scholar, 41.Freund C. Gehrig P. Holak T.A. Plückthun A. FEBS Lett. 1997; 407: 42-46Crossref PubMed Scopus (19) Google Scholar, 42.Jäger M. Plückthun A. FEBS Lett. 1997; 418: 106-110Crossref PubMed Scopus (39) Google Scholar). The rate-limiting step is the trans,cis-isomerization of the conserved Pro-L95 in the variable κ domain, which is a necessary prerequisite for the correct docking of VH. Human cyclophilin and E. coli PpiA have been shown to catalyze this reaction in vitro for the isolated VL domain and the Fv fragment (where the domains are unlinked) (42.Jäger M. Plückthun A. FEBS Lett. 1997; 418: 106-110Crossref PubMed Scopus (39) Google Scholar), but inefficiently or even not at all for scFv fragments, probably because the domains are inaccessible in an early folding intermediate (36.Jäger M. Plückthun A. J. Mol. Biol. 1999; 285: 2005-2019Crossref PubMed Scopus (42) Google Scholar, 41.Freund C. Gehrig P. Holak T.A. Plückthun A. FEBS Lett. 1997; 407: 42-46Crossref PubMed Scopus (19) Google Scholar, 42.Jäger M. Plückthun A. FEBS Lett. 1997; 418: 106-110Crossref PubMed Scopus (39) Google Scholar). The slow step of the scFv fragment of the antibody hu4D5-8, with or without disulfides, cannot be accelerated by PPIases (neither PpiA2 nor FkpA) (35.Ramm K. Plückthun A. J. Biol. Chem. 2000; 275: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).FkpA has been independently selected on two occasions with different antibodies. It is remarkable that it also dramatically improves the folding of an antibody whose rate-limiting step it cannot catalyzein vitro (4D5− −), and even more striking is that its coexpression increases the yield of an scFv fragment lackingcis-prolines. This suggests a folding assisting activity that is independent of the PPIase activity. This result is supported by the fact that neither PpiA nor SurA can replace FkpA in coexpression experiments. Previous experiments (9.Knappik A. Krebber C. Plückthun A. Bio/Technology. 1993; 11: 77-83Crossref PubMed Scopus (13) Google Scholar) in which PpiA was overexpressed together with the Fv, scFv, and Fab fragments of the antibody McPC603 also failed to show any significant effect for any of the fragments, consistent with the results found here for PpiA with a different series of scFv fragments.A “chaperone-like” activity of PPIases has been discussed controversially before (43.Freskgård P.O. Bergenhem N. Bengt-Harald J. Svensson M. Carlsson U. Science. 1992; 258: 466-468Crossref PubMed Scopus (185) Google Scholar, 44.Rinfret A. Collins C. Ménard R. Anderson S.K. Biochemistry. 1994; 33: 1668-1673Crossref PubMed Scopus (46) Google Scholar, 45.Kern G. Kern D. Schmid F.X. Fischer G. FEBS Lett. 1994; 348: 145-148Crossref PubMed Scopus (65) Google Scholar). The debate was fueled by the fact that the model system used, human carbonic anhydrase II, still contains twocis-prolines, requiring complicated analyses. Experiments with citrate synthase as substrate showed that FKBP52 possesses chaperone activity independently of its PPIase activity (46.Bose S. Weikl T. Bügl H. Buchner J. Science. 1996; 274: 1715-1717Crossref PubMed Scopus (316) Google Scholar). Additionally, cyclophilin Cyp-40 shows chaperone activity on denatured β-galactosidase, which is not affected by cyclosporin A, suggesting that its chaperone activity is not dependent on peptidylcis,trans-isomerase activity (47.Freeman B.C. Toft D.O. Morimoto R.I. Science. 1996; 274: 1718-1720Crossref PubMed Scopus (288) Google Scholar).Function and Regulation of FkpAIn E. coli, all periplasmic activities of FkpA, PpiA, SurA, and PpiD have been individually deleted, and the cells remained viable, also in the double mutants surA/rotA and fkpA/rotA (21.Missiakas D. Betton J.M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar, 48.Kleerebezem M. Heutink M. Tommassen J. Mol. Microbiol. 1995; 18: 313-320Crossref PubMed Scopus (33) Google Scholar, 49.Dartigalongue C. Raina S. EMBO J. 1998; 17: 3968-3980Crossref PubMed Scopus (188) Google Scholar). Nevertheless, based on the effects seen with surA mutants, SurA has been proposed to be involved in outer membrane protein localization and folding (50.Lazar S.W. Kolter R. J. Bacteriol. 1996; 178: 1770-1773Crossref PubMed Google Scholar, 51.Rouvière P.E. Gross C.A. Genes Dev. 1996; 10: 3170-3182Crossref PubMed Scopus (247) Google Scholar), as has PpiD (49.Dartigalongue C. Raina S. EMBO J. 1998; 17: 3968-3980Crossref PubMed Scopus (188) Google Scholar). Interestingly, thesurA/ppiD double mutant causes lethality, whereas thesurA null mutant can be combined with ppiA,fkpA, or even an skp null mutant. No particular substrate protein has been proposed yet for PpiA or FkpA.Although the deletion of FkpA is not lethal, it leads to increased ςE activity (40.Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (209) Google Scholar). Together with the findings that high level synthesis of FkpA can suppress the accumulation of unfolded periplasmic and outer membrane proteins, it has been suggested that it acts as a global folding catalyst (21.Missiakas D. Betton J.M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar). The expression of FkpA is increased by overproduction of ςE and by creation of extracytoplasmic stress; additionally, ςE-binding sites are found in its promoter region (40.Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (209) Google Scholar).Our experiments do not suggest that the physiological role of FkpA forE. coli periplasmic proteins is independent of its PPIase activity, which it unequivocally has (35.Ramm K. Plückthun A. J. Biol. Chem. 2000; 275: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). On the other hand, the repeated enrichment of FkpA in independent phage panning experiments shows the strong benefit of FkpA overexpression, which is more strikingly verified with model proteins not containingcis-prolines. Were the effect “unspecific,” a wide variety of proteins should be able to improve antibody expression when overexpressed. This is, however, not the case.It would be plausible to propose that the peptide-binding site of FkpA may be used to reversibly bind part of the antibody. Whether FkpA acts as a “protein buffer” (52.Rutherford S.L. Lindquist S. Nature. 1998; 396: 336-342Crossref PubMed Scopus (1700) Google Scholar); binds early in the pathway like the trigger factor, which is a ribosome-bound FKBP (53.Valent Q.A. Kendall D.A. High S. Kusters R. Oudega B. Luirink J. EMBO J. 1995; 14: 5494-5505Crossref PubMed Scopus (238) Google Scholar, 54.Callebaut I. Mormon J.P. FEBS Lett. 1995; 374: 211-215Crossref PubMed Scopus (70) Google Scholar, 55.Hesterkamp T. Hauser S. Lütcke H. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4437-4441Crossref PubMed Scopus (203) Google Scholar); rescues proteins that have denatured from the native state (56.Mayer M.P. Bukau B. Curr. Biol. 1999; 9: R322-R325Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar); or does even all of the above remains the subject of further studies.Multiple Functions of PPIasesThe dual biological role of these classes of proteins, which, on the one hand, are able to catalyze the isomerization of a peptide bond and, on the other hand, are involved in signal transduction at least in some cells (57.Jayaraman T. Brillantes A.M. Timerman A.P. Fleischer S. Erdjument-Bromage H. Tempst P. Marks A.R. J. Biol. Chem. 1992; 267: 9474-9477Abstract Full Text PDF PubMed Google Scholar, 58.Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 59.Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar, 60.Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar, 61.Melnick J. Argon Y. Immunol. Today. 1995; 16: 243-250Abstract Full Text PDF PubMed Scopus (73) Google Scholar), has never been fully resolved. Particularly, it has remained mysterious why so many apparently redundant activities are present in most genomes. Moreover, attempts to delete one or several of these enzymes have rarely led to obvious phenotypic effects. In addition to the experiments performed with the deletion of the E. coli genes (see above), all 12 cyclophilin and FKBP genes of yeast, identified with the knowledge of the complete genome sequence, have been deleted, but the cells were still viable (62.Dolinski K. Muir S. Cardenas M. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13093-13098Crossref PubMed Scopus (249) Google Scholar), whereas the deletion of the only parvulin homologue in yeast, ESS1, is lethal (63.Rahfeld J.U. Rücknagel K.P. Schelbert B. Ludwig B. Hacker J. Mann K. Fischer G. FEBS Lett. 1994; 352: 180-184Crossref PubMed Scopus (170) Google Scholar, 64.Hani J. Stumpf G. Domdey H. FEBS Lett. 1995; 365: 198-202Crossref PubMed Scopus (83) Google Scholar). Similarly, two out of four PPIases have been deleted fromBacillus subtilis, with no phenotypic consequence in rich medium or under several stress conditions tested. The double mutant showed a strongly retarded growth behavior only in the absence of all amino acids except tryptophan and phenylalanine (65.Göthel S.F. Scholz C. Schmid F.X. Marahiel M.A. Biochemistry. 1998; 37: 13392-13399Crossref PubMed Scopus (63) Google Scholar). In Erwinia chrysanthemi, disruption of rotA caused no change in cell morphology, cell viability, growth rate, or stability of the extracellular and periplasmic proteins (66.Pissavin C. Hugouvieux-Cotte-Pattat N. FEMS Microbiol. Lett. 1997; 157: 59-65Crossref PubMed Google Scholar). It is certainly possible that a subset of PPIases fulfills a more general binding function, which can be exploited to improve folding.ConclusionsTaking all the facts together, we conclude that the beneficial effect of FkpA overexpression must be independent of proline cis,trans-isomerase activity. First, the effect is found only with FkpA, but not with PpiA or SurA; and second, it is found also with a model protein devoid of any cis-prolines. Additionally, a very strong effect is found with a protein (4D5− −) whose rate-limiting step cannot be accelerated by FkpA (35.Ramm K. Plückthun A. J. Biol. Chem. 2000; 275: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These in vivo findings are directly reflectedin vitro, where the presence of FkpA during folding improves the yield under conditions favoring aggregation, but does not accelerate the slow step (35.Ramm K. Plückthun A. J. Biol. Chem. 2000; 275: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Our results have also validated the selection strategy that uses phage display of a poorly expressible protein. It is likely that this approach can be repeated, even with libraries of genes from organisms other than E. coli. Finally, the overexpression of FkpA helps to solve a long-standing practical problem in antibody engineering by improving the expression of a wide variety of different antibodies tested. An important strategy for the production of recombinant disulfide-containing proteins is their secretion to the periplasmic space of Escherichia coli, as the periplasm is the location of the disulfide-forming machinery (1.Bardwell J.C. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (197) Google Scholar, 2.Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar). Antibodies are one class of particularly important disulfide-containing proteins that have frequently been expressed in this compartment, and this is probably the most convenient method to obtain engineered antibodies for research and medical or technological applications (3.Plückthun A. Krebber A. Krebber C. Horn U. Knüpfer R. Wenderoth L. Nieba L. Proba K. Riesanberg D. Antibody Engineering: A Practical Approach. IRL Press Ltd., Oxford1996: 203-252Google Scholar, 4.Huston J.S. McCartney J. Tai M.S. Mottola-Hartshorn C. Jin D. Warren F. Keck P. Oppermann H. Int. Rev. Immunol. 1993; 10: 195-217Crossref PubMed Scopus (150) Google Scholar, 5.Dall'Acqua W. Carter P. Curr. Opin. Struct. Biol. 1998; 8: 443-450Crossref PubMed Scopus (87) Google Scholar). Unfortunately, folding problems of antibodies and other heterologous proteins in the periplasm are frequently observed, and the nature and existence of molecular chaperones in this compartment are still only poorly understood (2.Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (237) Google Scholar, 6.Wall J.G. Plückthun A. Curr. Opin. Biotechnol. 1995; 6: 507-516Crossref PubMed Scopus (81) Google Scholar, 7.Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (187) Google Scholar). It has been previously shown (8.Skerra A. Plückthun A. Protein Eng. 1991; 4: 971-979Crossref PubMed Scopus (90) Google Scholar, 9.Knappik A. Krebber C. Plückthun A. Bio/Technology. 1993; 11: 77-83Crossref PubMed Scopus (13) Google Scholar, 10.Knappik A. Plückthun A. Protein Eng. 1995; 8: 81-89Crossref PubMed Scopus (218) Google Scholar, 11.Ulrich H.D. Patten P.A. Yang P.L. Romesberg F.E. Schultz P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11907-11911Crossref PubMed Scopus (84) Google Scholar, 12.Forsberg G. Forsgren M. Jaki M. Norin M. Sterky C. Enhorning A. Larsson K. Ericsson M. Björk P. J. Biol. Chem. 1997; 272: 12430-12436Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) that the functional periplasmic expression of antibody fragments can be limited by the sequence-dependent, moderate efficiency of periplasmic folding, which can furthermore be accompanied by cell lysis, as has also been observed for other proteins (13.Suominen I. Karp M. Lahde M. Kopio A. Glumoff T. Meyer P. Mantsala P. Gene (Amst.). 1987; 61: 165-176Crossref PubMed Scopus (44) Google Scholar). Typically, the actual membrane transport is not limiting (8.Skerra A. Plückthun A. Protein Eng. 1991; 4: 971-979Crossref PubMed Scopus (90) Google Scholar), and large amounts of correctly processed, but precipitated protein are available in the periplasm. It is still unclear which factors, if any, might play a role in preventing the aggregation and in guiding the correct folding of functional antibody fragments or other periplasmic proteins in E. coli, and it is certainly conceivable that different factors play a role for different proteins. We have recently developed a selection system for the identification of novel factors that may help the functional periplasmic expression of a substrate protein in question (14.Bothmann H. Plückthun A. Nature Biotechnol. 1998; 16: 376-380Crossref PubMed Scopus (163) Google Scholar). It was based on the assumption that the folding of soluble periplasmic protein occurs in contact with the same machinery and in the same environment as that of a protein displayed on filamentous phage. Fusion proteins with the minor coat protein gene-3 protein (g3p)1of filamentous phage are transiently produced in a state anchored to the inner membrane, before they are incorporated into the phage coat (15.Davis N.G. Boeke J.D. Model P. J. Mol. Biol. 1985; 181: 111-121Crossref PubMed Scopus (87) Google Scholar). Thus, the folding of the fusion protein occurs in the periplasm and should be influenced by the same factors as that of a soluble periplasmic protein. The selection system works by displaying a poorly expressing antibody fragment on filamentous phage and coexpressing a library of E. coli genes on the same phagemid. Note that these E. coli proteins encoded by the library are not displayed; they are merely expressed by the host cell producing a particular phage in question. If the coexpressed factor improves expression of the antibody-g3p fusion protein, a higher percentage of the phage particles will carry a functional antibody fragment and thus be selectable, even though this displayed antibody fragment is identical in all cases. In a previous study, we identified the periplasmic protein Skp (OmpH, HlpA) by this methodology (14.Bothmann H. Plückthun A. Nature Biotechnol. 1998; 16: 376-380Crossref PubMed Scopus (163) Google Scholar). Since we wanted to select for additional factors that might increase the expression yield, we have repeated this selection in the present study, this time using anE. coli library prepared from a strain devoid of theskp gene. We enriched the gene coding for the protein FkpA, a periplasmic peptidylprolyl cis,trans-isomerase (16.Horne S.M. Young K.D. Arch. Microbiol. 1995; 163: 357-365Crossref PubMed Scopus (84) Google Scholar). Upon characterizing the effect further, we found that only FkpA, but not the other periplasmic peptidylprolyl cis,trans-isomerases PpiA and SurA, had any such effect, suggesting that there is no functional redundancy in these enzymes. Most intriguingly, we found that FkpA has also a very beneficial effect on antibody fragments whose rate-limiting proline isomerization it cannot catalyze and, particularly noteworthy, that do not have any cis-proline at all. DISCUSSIONIn this work, we validated our selection system for periplasmic folding factors. They may be extremely useful in increasing the functional yield of recombinant proteins such as antibodies, which need to be produced in the periplasm because of their disulfide bonds. We believe that this selection approach is very promising for the identification of additional factors involved in periplasmic folding and in preventing misfolding and that this strategy can be expanded to the screening of different genetic libraries for such factors. The protein FkpA, which we have enriched, indeed clearly improves the expression yield of a wide range of functional scFv fragments in the periplasm of E. coli (in some cases, 10-fold). This greatly beneficial effect on the folding yield is visible for all scFv fragments tested, except one, which has good folding properties anyway.FkpA was first described as an E. coli protein homologous to the eukaryotic FK506-binding proteins (FKBPs) (16.Horne S.M. Young K.D. Arch. Microbiol. 1995; 163: 357-365Crossref PubMed Scopus (84) Google Scholar), a class of well characterized PPIases that have been shown to be inhibitable by the macrolide FK506. Mature FkpA is located in the periplasm (21.Missiakas D. Betton J.M. Raina S. Mol. Microbiol. 1996; 21: 871-884Crossref PubMed Scopus (292) Google Scholar), and its gene carries ςE-binding sites in the promoter" @default.
W1989287988 created "2016-06-24" @default.
W1989287988 creator A5083658123 @default.
W1989287988 creator A5089656814 @default.
W1989287988 date "2000-06-01" @default.
W1989287988 modified "2023-10-14" @default.
W1989287988 title "The Periplasmic Escherichia coli Peptidylprolyl cis,trans-Isomerase FkpA" @default.
W1989287988 cites W1545713086 @default.
W1989287988 cites W1549665441 @default.
W1989287988 cites W1550809462 @default.
W1989287988 cites W1554180547 @default.
W1989287988 cites W1554245708 @default.
W1989287988 cites W1607954709 @default.
W1989287988 cites W1754378627 @default.
W1989287988 cites W1973974738 @default.
W1989287988 cites W1975237639 @default.
W1989287988 cites W1980894249 @default.
W1989287988 cites W1982664434 @default.
W1989287988 cites W1983314769 @default.
W1989287988 cites W1987823655 @default.
W1989287988 cites W1989233913 @default.
W1989287988 cites W1990279175 @default.
W1989287988 cites W1993353096 @default.
W1989287988 cites W1997352972 @default.
W1989287988 cites W1997540061 @default.
W1989287988 cites W1997830689 @default.
W1989287988 cites W1997841502 @default.
W1989287988 cites W1999465079 @default.
W1989287988 cites W2005174079 @default.
W1989287988 cites W2006174330 @default.
W1989287988 cites W2009376936 @default.
W1989287988 cites W2009391528 @default.
W1989287988 cites W2018283368 @default.
W1989287988 cites W2026448162 @default.
W1989287988 cites W2035250546 @default.
W1989287988 cites W2040331602 @default.
W1989287988 cites W2044784863 @default.
W1989287988 cites W2046439007 @default.
W1989287988 cites W2048600363 @default.
W1989287988 cites W2050942714 @default.
W1989287988 cites W2054774755 @default.
W1989287988 cites W2056374924 @default.
W1989287988 cites W2057547483 @default.
W1989287988 cites W2059568862 @default.
W1989287988 cites W2059998232 @default.
W1989287988 cites W2065931405 @default.
W1989287988 cites W2079336387 @default.
W1989287988 cites W2080394439 @default.
W1989287988 cites W2104098314 @default.
W1989287988 cites W2107297174 @default.
W1989287988 cites W2112922048 @default.
W1989287988 cites W2118888416 @default.
W1989287988 cites W2118991108 @default.
W1989287988 cites W2119416327 @default.
W1989287988 cites W2126664074 @default.
W1989287988 cites W2130930583 @default.
W1989287988 cites W2141385342 @default.
W1989287988 cites W2143703414 @default.
W1989287988 cites W2145780697 @default.
W1989287988 cites W2152135678 @default.
W1989287988 cites W2157194928 @default.
W1989287988 cites W2158078460 @default.
W1989287988 cites W2158196711 @default.
W1989287988 cites W2161243122 @default.
W1989287988 cites W2165658854 @default.
W1989287988 cites W2169769186 @default.
W1989287988 cites W2171959398 @default.
W1989287988 cites W2347230326 @default.
W1989287988 cites W4241225489 @default.
W1989287988 doi "https://doi.org/10.1074/jbc.m910233199" @default.
W1989287988 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10748200" @default.
W1989287988 hasPublicationYear "2000" @default.
W1989287988 type Work @default.
W1989287988 sameAs 1989287988 @default.
W1989287988 citedByCount "165" @default.
W1989287988 countsByYear W19892879882012 @default.
W1989287988 countsByYear W19892879882013 @default.
W1989287988 countsByYear W19892879882014 @default.
W1989287988 countsByYear W19892879882015 @default.
W1989287988 countsByYear W19892879882016 @default.
W1989287988 countsByYear W19892879882017 @default.
W1989287988 countsByYear W19892879882018 @default.
W1989287988 countsByYear W19892879882019 @default.
W1989287988 countsByYear W19892879882020 @default.
W1989287988 countsByYear W19892879882021 @default.
W1989287988 countsByYear W19892879882022 @default.
W1989287988 countsByYear W19892879882023 @default.
W1989287988 crossrefType "journal-article" @default.
W1989287988 hasAuthorship W1989287988A5083658123 @default.
W1989287988 hasAuthorship W1989287988A5089656814 @default.
W1989287988 hasBestOaLocation W19892879881 @default.
W1989287988 hasConcept C104317684 @default.
W1989287988 hasConcept C181199279 @default.
W1989287988 hasConcept C185592680 @default.
W1989287988 hasConcept C201663137 @default.
W1989287988 hasConcept C2781264208 @default.
W1989287988 hasConcept C2908821069 @default.
W1989287988 hasConcept C2910190745 @default.