Biotechnology Letters

, 31:1661 | Cite as

Extracellular recombinant protein production from Escherichia coli

Original Research Paper


Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.


Autotransporter system Kill proteins Lysis-promoting proteins Protein secretion Secretion systems Secretome 


  1. Angkawidjaja C, Kuwahara K, Omori K, Koga Y, Takano K, Kanaya S (2006) Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization. Protein Eng Des Sel 19:337–343PubMedCrossRefGoogle Scholar
  2. Beshay U, Miksch G, Friehs K, Flaschel E (2007) Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter. Biotechnol Lett 29:1893–1901PubMedCrossRefGoogle Scholar
  3. Beshay U, Miksch G, Friehs K, Flaschel E (2009) Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli. Bioprocess Biosyst Eng 32:149–158PubMedCrossRefGoogle Scholar
  4. Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635PubMedCrossRefGoogle Scholar
  5. Chung CW, You J, Kim K, Moon Y, Kim H, Ahn JH (2009) Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD). Microb Cell Fact 8:1–11CrossRefGoogle Scholar
  6. Cols N, Roepatorff K, Gonzalez-Duarte R, Atrian S (2001) Secretion of mouse-metallothionein by engineered E. coli cells in metal-enriched culture media. J Mol Microbiol Biotechnol 3:507–512PubMedGoogle Scholar
  7. Desvaux M, Parham NJ, Henderson IR (2004) The autotransporter secretion system. Res Microbiol 155:53–60PubMedCrossRefGoogle Scholar
  8. Dresler K, van den Heuvel J, Müller RJ, Deckwer WD (2006) Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli. Bioprocess Biosyst Eng 29:169–183PubMedCrossRefGoogle Scholar
  9. Fernandez LA (2004) Prokaryotic expression of antibodies and affibodies. Curr Opin Biotechnol 15:364–373PubMedCrossRefGoogle Scholar
  10. Filloux A (2004) The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694:163–179PubMedCrossRefGoogle Scholar
  11. Francetic O, Belin D, Badaut C, Pugsley AP (2000) Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. EMBO J 19:6697–6703PubMedCrossRefGoogle Scholar
  12. Fu Z, Hamid SA, Razak CAN, Basri M, Salleh AB, Rahman R (2003) Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Protein Expr Purif 28:63–68PubMedCrossRefGoogle Scholar
  13. Gentschev I, Mollenkopf H, Sokolovic Z, Hess J, Kaufmann SHR, Goebel W (1996) Development of antigen-delivery systems based on the Escherichia coli hemolysin secretion pathway. Gene 179:133–140PubMedCrossRefGoogle Scholar
  14. Gumpert J, Hoischen C (1998) Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products. Curr Opin Biotechnol 9:506–509PubMedCrossRefGoogle Scholar
  15. Gupta P, Lee KH (2008) Silent mutations result in HlyA hypersecretion by reducing intracellular HlyA protein aggregates. Biotechnol Bioeng 101:967–974PubMedCrossRefGoogle Scholar
  16. Holland IB, Schmitt L, Young J (2005) Type I protein secretion in bacteria, the ABC-transporter dependent pathway. Mol Membr Biol 22:29–39PubMedCrossRefGoogle Scholar
  17. Ignatova Z, Mahsunah A, Georgieva M, Kasche V (2003) Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains. Appl Environ Microbiol 69:1237–1245PubMedCrossRefGoogle Scholar
  18. Jermy A (2009) Bacterial secretion: turning the cogs in type VI secretion. Nat Rev Microbiol 7:175–176CrossRefGoogle Scholar
  19. Khushoo A, Pal Y, Mukherjee KJ (2005) Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shaker-flask and bioreactor. Appl Microbiol Biotechnol 68:189–197PubMedCrossRefGoogle Scholar
  20. Kim CK, Lee SY, Kwon OJ, Lee SM, Nah SY, Jeong SM (2007) Secretory expression of active clostripain in Escherichia coli. J Biotechnol 131:346–352PubMedCrossRefGoogle Scholar
  21. Kitai K, Kudo T, Nakamura S, Masegi T, Ichikawa Y (1988) Extracellular production of human immunoglobulin region (hIgG-Fc) by Escherichia coli. Appl Microbiol Biotechnol 28:52–56CrossRefGoogle Scholar
  22. Langer ES (2009) Capitalizing on novel expression systems. Genet Eng Biotechnol News 15:12Google Scholar
  23. Lee PS, Lee KH (2004) Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses. Biotechnol Bioeng 89:195–205CrossRefGoogle Scholar
  24. Lee J, Sarawat V, Koh I, Song K, Park Y, Rhee S (2001) Secretory production of arthrobacter levan fructotransferase from recombinant Escherichia coli. FEMS Microbiol Lett 193:127–133CrossRefGoogle Scholar
  25. Lindeberg M, Salmond GPC, Collmer A (1996) Complementation of deletion mutations in a cloned functional cluster Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mol Microbiol 20:175–190PubMedCrossRefGoogle Scholar
  26. Lurink J, Watnabe T, Wu HC, Stegehuis F, De Graaf FK, De Graaf BO (1987) Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of b-lactamase. J Bacteriol 169:2345–2350Google Scholar
  27. Majander K, Anto L, antikainen J, Lang H, Brummer M, Korhnnen TK, Westerlund-Wilstronm B (2005) Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol 23:475–481PubMedCrossRefGoogle Scholar
  28. Miksch G, Ryu S, Risse JM, Flaschel E (2008) Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein. Appl Microbiol Biotechnol 81:319–326PubMedCrossRefGoogle Scholar
  29. Mota LJ, Cornelis GR (2005) The bacterial injection kit: type III secretion systems. Ann Med 37:234–249PubMedCrossRefGoogle Scholar
  30. Nandakumar MP, Cheung A, Marten MR (2006) Proteomic analysis of extracellular proteins from Escherichia coli W3110. J Proteome Res 5:1155–1161PubMedCrossRefGoogle Scholar
  31. Ni Y, Reye J, Chen RR (2007) lpp deletion as a permeabilization method. Biotechnol Bioeng 97:1347–1356PubMedCrossRefGoogle Scholar
  32. Nisole A, Lussier F, Morley KL, Shareck F, Kazlauskas RJ, Dupont C, Pelletier JN (2006) Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity. Protein Expr Purif 46:274–284PubMedCrossRefGoogle Scholar
  33. Papi RM, Shaitidou SA, Trikka FA, Kyriakidis DA (2005) Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase. Microb Cell Fact 4:35PubMedCrossRefGoogle Scholar
  34. Parente D, Raucci G, D’Alatri L, D’Estais G, Novelli S, Pacilli A, Saccinto MP, Mele A, De Santis R (1998) Overproduction of soluble extracellular cytotoxin α-Sarcin in Escherichia coli. Mol Biotechnol 9:99–106PubMedCrossRefGoogle Scholar
  35. Park SW, Chung MG, Lee HY, Kim JY, Rhee YH (2008) Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli. J Microbiol 46:662–669PubMedCrossRefGoogle Scholar
  36. Qian Z, Xia X, Choi JH, Lee SY (2008) Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnol Bioeng 101:587–601PubMedCrossRefGoogle Scholar
  37. Rahman R, Leow TC, Basri M, Salleh AB (2005) Secretory expression of thermostable T1 lipase through bacteriocin release protein. Protein Expr Purif 40:411–416PubMedCrossRefGoogle Scholar
  38. Rinas U, Hoffmann F (2004) Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli. Biotechnol Prog 20:679–687PubMedCrossRefGoogle Scholar
  39. 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):481–486PubMedCrossRefGoogle Scholar
  40. Shin H, Chen RR (2008) Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant. Biotechnol Bioeng 101:1288–1296PubMedCrossRefGoogle Scholar
  41. Wai SN, Lindmark B, Soderblom E, Takade M, Wetermark M, Oscarsson J, Jass A, Richter-Dahlfors A, Mizuoe Y, Uhlin BE (2003) Vesicle-mediated export and assembly of pore-forming oligomers of enterobacterial ClyA cytotoxin. Cell 115:25–35PubMedCrossRefGoogle Scholar
  42. Xia X, Han M, Lee SY, Yoo J (2008) Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation. Proteomics 8:2089–2103PubMedCrossRefGoogle Scholar
  43. Zhang G, Brokx S, Weiner JH (2006) Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nat Biotechnol 24:100–104PubMedCrossRefGoogle Scholar
  44. 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 65:2439–2445PubMedGoogle Scholar
  45. Zhu C, Ruiz-Perez F, Yan Z, Mao Y, Hackethal VL, Greco KM, Choy W, Davis K, Butteron JR, Boedeker EC (2006) Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli. Vaccine 24:3821–3831PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  2. 2.School of BiotechnologyJiangnan UniversityWuxiChina
  3. 3.School of Chemical and Biomolecular EngineeringGeorgia Institute of TechnologyAtlantaUSA

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