Abstract
The functional analysis of individual proteins or of multiprotein complexes—since the completion of several genome sequencing projects—is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.
Similar content being viewed by others
References
Ades SE, Connolly LE, Alba BM, Gross CA (1999) The Escherichia coli sigma(E)-dependent extracytoplasmic stress response is controlled by the regulated proteolysis of an anti-sigma factor. Genes Dev 13:2449–2461
Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408
Bardwell JCA, Lee J-O, Jander G, Martin N, Belin D, Beckwith JA (1993) Pathway for Disulfide Bond Formation in vivo. Proc Natl Acad Sci USA 90:1038–1042
Blondel A, Bedouelle H (1990) Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli. Eur J Biochem 193:325–330
Bothmann H, Plückthun A (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat Biotechnol 16:376–380
Chen R, Henning U (1996) A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins. Mol Microbiol 19(6):1287–1294
Connelly L, De Las Penas A, Alba BM, Gross CA (1997) The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways. Genes Dev 11:2012–2021
Danese PN, Silhavy TJ (1997) The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. Genes Dev 11:1183–1193
Danese PN, Snyder WB, Cosma CL, Davis LJ, Silhavy TJ (1995) The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease DegP. Genes Dev 9:387–398
Dartigalongue C, Raina S (1998) A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli. EMBO J 17:3968–3980
Elleby B, Svensson S, Wu X, Stefansson K, Nilsson J, Hallén D, Oppermann U, Abrahmsén L (2004) High-level production and optimization of monodispersity of 11β-hydroxysteriod dehydrogenase type 1. Biochem Biophys Acta 1700:199–207
Georgiou G, Segatori L (2005) Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr Opin Biotechnol 16:538–545
Gottesman S, Wickner S, Maurizi MR (1997) Protein quality control: triage by chaperones and proteases. Genes Dev 11:815–823
Horn U, Strittmatter W, Krebber A, Knüpfer U, Kujau M, Wenderoth R, Müller K, Matzku S, Plückthun A, Riesenberg D (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli using an optimized expression vector and high cell density fermentation under non-limiting growth conditions. Appl Microbiol Biotechnol 46:524–532
Hsiung HM, Cantrell A, Luirink J, Oudega B, Veros AJ, Becker GW (1989) Use of bacteriocin release protein in E. coli for excretion of human growth hormone into the culture medium. Bio/Technology 7:267–271
Kraft M, Knüpfer U, Wenderoth R, Pietschmann P, Hock B, Horn U (2007) An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli. Appl Microbiol Biotechnol 75:397–406
Krebber A, Burmester J, Plückthun A (1996) Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 178:71–74
Larsen JEL, Gerdes K, Light J, Molin S (1984) Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter. Gene 28:45–54
LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11:187–193
Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, Honegger A, Klinger B, Mocikat R, Plückthun A (1997) Specific detection of His–tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. Biotechniques 22:140–149
Lipinska B, Sharma S, Georgopoulos C (1988) Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-independent mechanism of heat-inducible transcription. Nucl Acids Res 16:10053–10067
Lippert K, Galinski EA (1992) Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol 37:61–65
Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538
Min KT, Kim MH, Lee DS (1988) Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis. Nucleic Acids Res 16:5075–5088
Missiakas D, Betton JM, Raina S (1996) New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol Microbiol 21:871–884
Monteilhet C, Lachacinski N, Aggerbeck LP (1993) Cytoplasmic and periplasmic production of human apolipoprotein-E in Escherichia coli using natural and bacterial signal peptide. Gene 125:223–228
Nieba-Axmann SE, Plückthun A (1997) Chaperone-mediated protein folding. Bioforum Int 1:20–25
Nordström K, Molin S, Aagaard-Hansen H (1980a) Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region. Plasmid 4:215–227
Nordström K, Molin S, Aagaard-Hansen H (1980b) Partitioning of plasmid R1 in Escherichia coli. II. Incompatibility properties of the partitioning system. Plasmid 4:332–339
Pack P, Plückthun A (1992) Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 31:1579–1584
Plückthun A (1998) Studying protein structure and function by directed evolution: examples with engineered antibodies, protein dynamics, function and design. In: Jardetzky O, LeFevre JF (eds) NATO ASI series, Series A (Life Sciences vol. 301), Plenum, New York
Plückthun A, Krebber A, Krebber C, Horn U, Knüpfer U, Wenderoth R, Nieba L, Proba K, Riesenberg D (1996) Producing antibodies in Escherichia coli: From PCR to fermentation, in “Antibody Engineering, A practical approach”, IRL, Oxford, pp 203–252
Pogliano J, Lynch AS, Belin D, Lin EC, Beckwith J (1997) Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev 11:1169–1182
Raffa RG, Raivio TL (2002) A third envelope stress signal transduction pathway in Escherichia coli. Mol Microbiol 45:1599–1611
Raina S, Missiakas D, Georgopoulos C (1995) The rpoE gene encoding the sigma E (sigma 24) heat shock sigma factor of Escherichia coli. EMBO J 14:1043–1055
Raivio TL, Silhavy TJ (1999) The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses. Curr Opin Microbiol 2:159–165
Robinson RR, Liu AY, Horowitz H, Better M, Wall R, Lei SP, Wilcox GL (1998) Pectate lyase signal sequence. Patent US 5846818
Rouviere PE, De Las Penas A, Mecsas J, Lu CZ, Rudd KE, Gross CA (1995) rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli. EMBO J 14:1032–1042
Schlapschy M, Grimm S, Skerra A (2006) A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli. Protein Eng Des Sel 19:385–390
Strauch KL, Johnson K, Beckwith J (1989) Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J Bacteriol 171:2689–2696
Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17:691–695
Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328:575–592
Walker EA, Clark AM, Hewison M, Ride JP, Steward PM (2001) Functional expression, characterization, and purification of the catalytic domain of human 11-β-hydroxysteriod dehydrogenase type 1. J Biol Chem 276:21343–21350
Wells A (1999) EGF receptor. Int J Biochem Cell Biol 31:637–643
Wigley WC, Stidham RD, Smith NM, Hunt JF, Thomas PJ (2001) Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat Biotechnol 19:131–136
Wimmer H, Olsson M, Petersen MT, Hatti-Kaul R, Peterson SB, Müller N (1997) Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol. J Biotechnol 55:85–100
Wörn A, Plückthun A (1998) Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochemistry 37:13120–13127
Xie G, Timasheff SN (1997) Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein. Protein Sci 6:211–221
Acknowledgments
We thank Andreas Plückthun for providing the plasmids pAK100, pHB110. Thanks to Gisela Sudermann and Liane Schlenckert for excellent technical support. Special thanks to Giles Johnson and Friedrich Kring for critical reading of the manuscript. This work was funded by Merck KGaA Darmstadt, Germany.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kraft, M., Knüpfer, U., Wenderoth, R. et al. A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli . Appl Microbiol Biotechnol 76, 1413–1422 (2007). https://doi.org/10.1007/s00253-007-1121-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-1121-7