Abstract
Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.
Similar content being viewed by others
References
Angelini S, Moreno R, Gouffi K, Santini C, Yamagishi A, Berenguer J, Wu L (2001) Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli. FEBS Lett 506:103-107
Arie J, Sassoon N, Betton J (2001) Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol Microbiol 39:199–210
Barrett CML, Ray N, Thomas JD, Robinson C, Bolhuis A (2003) Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli. Biochem Biophys Res Comm 304:279–284
Bothmann H, Pluckthun A (1998) Selection for a periplasmic factor improving phage display and periplasmic expression. Nat Biotechnol 16:376–380
Bothmann H, Pluckthun A (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. J Biol Chem 275:17100–17105
Chen G, Hayhurst A, Thomas JG, Garvey BR, Iverson BL, Georgiou G (2001) Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nat Biotechnol 19:537–542
Choi JH, Jeong KJ, Kim SC, Lee SY (2000) Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. Endoxylanase signal sequence. Appl Microbiol Biotechnol 53:640–645
Chowdhury PS, Kushwaha A, Abrol S, Chaudhary VK (1994) An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase. Protein Expr Purif 5:89–95
Chung BH, Sohn M, Oh S, Park U, Poo H, Kim BS, Yu MJ, Lee SK (1998) Overproduction of human granulocyte-colony stimulating factor fused to the PelB signal peptide in Escherichia coli. J Ferm Bioeng 85:443–446
Dracheva S, Palermo RE, Powers GD, Waugh DS (1995) Expression of soluble human interleukin-2 receptor α-chain in Escherichia coli. Protein Expr Purif 6:737–747
Fernandez LA, Sola I, Enjuanes L, De Lorenzo V (2000) Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system. Appl Environ Microbiol 66:5024–5029
Fu Z, Hamid SBA, Razak CAN, Basri M, Salleh AB, Rahman RNZA (2003) Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Protein Expr Purif 28:63–68
Guisez Y, Fache I, Campfield LA, Smith FJ, Farid A, Plaetinck G, Van der Heyden J, Tavernier J, Fiers W, Burn P, Devos R (1998) Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization. Protein Expr Purif 12:249–258
Holt RG, Raju L (2000) Signal sequence and alanine-rich region of streptococcal protein antigen A of Streptococcus sobrinus can direct localization of alkaline phosphatase to the periplasm of Escherichia coli. FEMS Microbiol Lett 184:17–21
Jang KH, Seo KB, Song KB, Kim CH, Rhee SK (1999) Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli. Bioproc Eng 21:453–458
Jeong KJ, Lee PC, Park IY, Kim MS, Kim SC (1998) Molecular cloning and characterization of an endoxylanase gene of Bacillus sp. in Escherichia coli. Enzyme Microb Technol 22(7):599–605
Jeong KJ, Lee SY (2000) Secretory production of human leptin in Escherichia coli. Biotechnol Bioeng 67:398-407
Jeong KJ, Lee SY (2001) Secretory production of human granulocyte colony-stimulating factor in Escherichia coli. Protein Expr Purif 23(2):311–8
Jeong KJ, Lee SY (2002) Excretory production of human β-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli. Appl Environ Microbiol 68:4979–4985
Jobling MG, Palmer LM, Erbe JL, Holmes RK (1997) Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli. Plasmid 38(3):158–73
Kaderbhai MA, Ugochukwu CC, Lamb DC, Kelly S (2000) Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli. Biochem Biophys Res Comm 279:803–807
Kaderbhai N, Karim A, Hankey W, Jenkins G, Venning J, Kaderbhai MA (1997) Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli. Biotechnol Appl Biochem 25:53–61
Kleist S, Miksch G, Hitzmann B, Arndt M, Friehs K, Flaschel E (2003) Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies. Appl Microbiol Biotechnol 61:456–462
Kujau MJ, Hoischen C, Riesenberg D, Gumpert J (1998) Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli: A comparison of the synthesis capacities of L-form strains with an E. coli producer strain. Appl Microbiol Biotechnol 49:51–58
Kurokawa Y, Yanagi H, Yura T (2001) Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli. J Biol Chem 276:14393–14399
Lam T, Wong RSC, Wong WR (1997) Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength. Enzyme Microb Technol 20:482-488
Lazar SW, Kolter R (1996) SurA assists the folding of Escherichia coli outer membrane proteins. J Bacteriol 178:1770–1773
Le Calvez H, Green JM, Baty D (1996) Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate PelB signal sequence. Gene 170:51–55
Lee J, Saraswat V, Koh I, Song KB, Park YH, Rhee SK (2001) Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli. FEMS Microbiol Lett 195(2):127-132
Lee SJ, Kim IC, Kim DM, Bae KH, Byun SM (1998) High level secetion of recombinant staphylokinase into periplasm of Escherichia coli. Biotechnol Lett 20:113–116
Lee SY (1996) High cell density cultivation of Escherichia coli. Trends Biotechnol 14:98–105
Li Y, Chen CX, von Specht B, Hahn HP (2002) Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli. Gene 25:437–447
Lin W, Huang S, Chou CP (2001a) DegP-coexpression minimizes inclusion-body formation upon overproduction of recombinant penicillin acylase in Escherichia coli. Biotechnol Bioeng 73:484–492
Lin W, Huang S, Chou CP (2001b) High-level extracellular production of penicillin acylase by genetic engineering of Escherichia coli. J Chem Technol Biotechnol 76:1030–1037
Loo T, Patchett ML, Norris GE, Lott JS (2002) Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F. Protein Expr Purif 24:90–98
Lucic MR, Forbes BE, Grosvenor SE, Carr JM, Wallace JC, Forsberg G (1998) Secretion in Escherichia coli and phage-display of recombinant insulin-like growth factor binding protein-2. J Biotechnol J Biotechnol 61:95–108
Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538
Matsumoto T, Katsura D, Kondo A, Fukuda H (2002) Efficient secretory overexpression of Bacillus subtilis pectate lyase in Escherichia coli and single-step purification. Biochem Eng J 12(3):175–179
Mergulhao FJM, Monteiro GA, Larsson G, Sanden AM, Farewell A, Nystrom T, Cabral JMS, Taipa MA (2003) Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB. Appl Microbiol Biotechnol 61:495–501
Miksch G, Neitzel R, Fiedler E, Friehs K, Flaschel E (1997) Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors. Appl Microbiol Biotechnol 47:120–126
Miksch G, Kleist S, Friehs K, Flaschel E (2002) Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol 56:685–694
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.
Nagahari K, Kanaya S, Munakata D, Aoyagi Y, Mizushima S (1985) Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human β-endorphin. EMBO J 16:3589–3592
Park SJ, Georgiou G, Lee SY (1999) Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli. Biotechnol Prog 15:164–167
Pillai D, Dixit A, Krishnan T, Garg LC (1996) Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence. Gene 173(2):271-274
Pratap J, Dikshit KL (1998) Efficient of signal peptide changes on the extracellular processing of streptokinase form Escherichia coli requirement for secondary structure at the cleavage junction. Mol Gen Genet 258:326–333
Pritchard MP, Ossetian R, Li DN, Henderson CJ, Burchell B, Wolf CR, Friedberg T (1997) A general strategy for the expression of recombinant human cytochrome P450 s in Escherichia coli using bacterial signal peptides: Expression of CYP3A4, CYP2A6, and CYP2E1. Arch Biochem Biophys 345:342–354
Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57:50–108
Pugsley AP, Bayan N, Sauvonnet N (2001) Disulfide bonds formation in secretion component PulK provides a possible explanation for the role of DsbA in pullulanase secretion. J Bacteriol 183:1312–1319
Qui J, Swartz JR, Georgiou G (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 64:4891–4896
Rathore D, Nayak SK, Batra JK (1997) Overproduction of fungal ribotoxin α-sarcin in Escherichia coli: generation of an active immunotoxin. Gene 190:31–35
Rippmann JF, Klein M, Hoischen C, Brocks B, Rettig WJ, Gumpert J, Pfizenmaier K, Mattes R, Moosmayer D (1998) Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl Environ Microbiol 64(12):4862-4869.
Robbens J, Raeymaekers A, Steidler L, Fiers W, Remaut E (1995) Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification. Protein Expr Purif 6(4):481-486
Rua ML, Atomi H, Schmidt-Dannert C, Schmid RD (1998) High-level expression of the thermoalkanophilic lipase from Bacillus thermocatenulatus in Escherichia coli. Appl Microbiol Biotechnol 49:405–410
Santini C, Bernadac A, Zhang M, Chanal A, Ize B, Blanco C, Wu L (2001) Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock. J Biol Chem 276:8159–8164
Shokri A, Saden AM and Larsson G (2003) Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbial Biotechnol 60:654–664
Sivakesava S, Xu ZN, Chen YH, Hackett J, Huang RC, Lam E, Lam TL, Siu KL, Wong RSC, Wong WKR (1999) Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system. Process Biochem 34:893–900
Sriubolmas N, Panbangred W, Sriurairatana S, Meevootisom V (1997) Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase. Appl Microbiol Biotechnol 47:373–378
Sroga GE, Dordick J (2002) A strategy for in vivo screening of subtilisin E reaction specificity in E. coli periplasm. Biotechnol Bioeng 78:761–769
Tan S, Wu W, Liu J, Kong Y, Pu Y, Yuan R (2002) Efficient expression and secretion of recombinant hirudin III in E. coli using the L-asparaginase II signal sequence. Protein Expr Purif 25:430-436
Tanaka T, Horio T, Matuo Y (2002) Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization. Biochem Biophys Res Commun, 295(1):163–6
Thomas JD, Daniel RA, Errington J, Robinson C (2001) Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol 39:47–53
Toksoy E, Onsan ZI, Kirdar B (2002) High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter. Appl Microbiol Biotechnol 59:239–245
Tong L, Lin Q, Wong WKR, Ali A, Lim D, Sung WL, Hew CL, Yang DSC (2000) Extracelllular expression, purification, and characterization of a winter flounder antifreeze polypeptide form Escherichia coli. Protein Expr Purif 18:175–181
Uchida H, Naito N, Asada N, Wada M, Ikeda M, Kobayashi H, Asanagi M, Mori K, Fujita Y, Konda K, Kusuhara N, Kamioka T, Nakashima K, Honjo M (1997) Secretion of authentic 20-kDa human growth hormone (20 K hGH) in Escherichia coli and properties of the purified product. J Biotechnol 55:101–112
Van der Wal FJ, ten Hagen-Jounman CM, Oudega B, Luirink J (1995) Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: extracellular production of the periplasmic molecular chaperone FaeE. Appl Microbiol Biotechnol 44:459–465
Van der Wal FJ, Koningstein G, ten Hagen CM, Oudega B, Luirink J (1998) Optimization of bacterocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-Derived BRP gene to uncouple lethality and quasi-lysis from protein release. Appl Environ Microbiol 64:392–398
Wan EW, Baneyx F (1998) TolAIII co-expression facilitates the recovery of periplasmic recombinant proteins into the growth medium of Escherichia coli. Protein Expr Purif 14:13–22
Winter J, Neubauer P, Glockshuber R, Rudolph R (2000) Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA. J Biotechnol 84(2):175-185
Wong WR, Ali AB, Ma MC (2003) Cloning, expression, and characterization of diuretic hormone Manduca diuresin from Manduca sexta in Escherichia coli. Protein Expr Purif 29:51–57
Wulfing C, Rappuoli R (1997) Efficient production of heat-labile endotoxin mutant proteins by overexpression of dabA in a degP-deficient Escherichia coli strain. Arch Microbiol 167:280–283
Xu R, Du P, Fan JJ, Zhang Q, Li TP, Gan RB (2002) High-level expression and secretion of recombinant mouse endostatin by Escherichia coli. Protein Expr Purif 24:453-459
Yang J, Moyana T, Mackenzie S, Xia Q and Xiang J (1998) One hundred Seventy-fold increase in excretion of an FV frangmant tumor necrosis factor alpha fusion protein (SFV/TNF-α) from Escherichia coli caused by the synergistic effects of glycine and triton X-100. Appl Environ Microbiol 64:2669–2874
Yim SC, Jeong KJ, Chang HN, Lee SY (2001) High level secretory production of human G-CSF by fed-batch culture of recombinant Escherichia coli. Bioproc Biosystems Eng 24:249–254
Zavialov AV, Batchikova NV, Korpela T, Petrovskaya LE, Korobko VG, Kersley J, MacIntyre S, Zavyalov S (2001) Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli. Appl Environ Microbiol 67:1805–1814
Zhou S, Yomano LP, Saleh AZ, Davis FC, Aldrich HC, Ingram LO (1999) Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B. Appl Environ Microbiol 65:2439–2445
Acknowledgements
This review was supported by the Korean Systems Biology Research Grant (M10309020000-03B5002-00000) from the Ministry of Science and Technology. Support from IBM through the IBM-SUR program is greatly appreciated.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Choi, J.H., Lee, S.Y. Secretory and extracellular production of recombinant proteins using Escherichia coli . Appl Microbiol Biotechnol 64, 625–635 (2004). https://doi.org/10.1007/s00253-004-1559-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-004-1559-9