Escherichia coli: The Leading Model for the Production of Recombinant Proteins

  • Paula Tucci
  • Victoria Veroli
  • Mario Señorale
  • Mónica Marín
Part of the Microorganisms for Sustainability book series (MICRO, volume 1)


Proteins are a diverse group of biomolecules that have many different biological activities and structures. Both in the industrial and research laboratory, proteins are used for very diverse purposes and are produced at different scales. Main fields of application of proteins are human and animal prophylactics, therapeutics, and diagnostics, as well as the food processing industry. Besides, proteins are employed as additives to generate a diverse range of products. Pectinases are employed in the food industry since 1930, in fruit juice manufacturing; other enzymes like papain, bromelain, pepsin, rennin, lipases, cellulases, and amylases have several industrial applications. Due to their relevance in medicine, proteins used as biopharmaceuticals are the best characterized. This chapter addresses the production of recombinant proteins as aligned with the concept of industrial sustainability and Escherichia coli as a model for heterologous protein production. It describes and updates the main production strategies and the current available tools for the protein expression system. The main disadvantages, as well as the improvements done to this model, are detailed. A final summary that includes industrial uses and fields of applications of recombinant proteins points out the relevance of this prokaryotic system.


Recombinant Protein Disulfide Bond Codon Usage Synonymous Codon Recombinant Protein Production 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was partially supported by Agencia Nacional de Investigación e Innovación (ANII, Uruguay) through Fondo María Viñas (FMV_3_2013_1_100859). Victoria Veroli was the recipient of fellowships from ANII (Uruguay).


  1. Arie JP, Miot M, Sassoon N, Betton JM (2006) Formation of active inclusion bodies in the periplasm of Escherichia coli. Mol Microbiol 62(2):427–437CrossRefPubMedGoogle Scholar
  2. Austin S, Nordstrom K (1990) Partition-mediated incompatibility of bacterial plasmids. Cell 60(3):351–354CrossRefPubMedGoogle Scholar
  3. Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Baeshen NA, Redwan EM (2015) Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J Microbiol Biotechnol 25(7):953–962CrossRefPubMedGoogle Scholar
  4. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408CrossRefPubMedGoogle Scholar
  5. Bayer ME, Bayer MH, Lunn CA, Pigiet V (1987) Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli. J Bacteriol 169(6):2659–2666CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bentley R, Meganathan R (1982) Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol Rev 46(3):241–280PubMedPubMedCentralGoogle Scholar
  7. Burgess RR (2009) Refolding solubilized inclusion body proteins. Methods Enzymol 463:259–282CrossRefPubMedGoogle Scholar
  8. Burgess RR, Deutscher MP (eds) (2009) Methods in enzymology. Guide to protein purification, vol 463. Methods Enzymol, 2009/11/07 edn. ElsevierGoogle Scholar
  9. Carson M, Johnson DH, McDonald H, Brouillette C, Delucas LJ (2007) His-tag impact on structure. Acta Crystallogr D Biol Crystallogr 63(Pt 3):295–301CrossRefPubMedGoogle Scholar
  10. Castillo V, Grana-Montes R, Sabate R, Ventura S (2011) Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes. Biotechnol J 6(6):674–685CrossRefPubMedGoogle Scholar
  11. Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64(5):625–635CrossRefPubMedGoogle Scholar
  12. Chow MK, Amin AA, Fulton KF, Fernando T, Kamau L, Batty C, Louca M, Ho S, Whisstock JC, Bottomley SP, Buckle AM (2006) The REFOLD database: a tool for the optimization of protein expression and refolding. Nucleic Acids Res 34(Database issue):D207–D212CrossRefPubMedGoogle Scholar
  13. Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6(6):715–730CrossRefPubMedGoogle Scholar
  14. Correa A, Oppezzo P (2015) Overcoming the solubility problem in E. coli: available approaches for recombinant protein production. Methods Mol Biol 1258:27–44CrossRefPubMedGoogle Scholar
  15. Cortazzo P, Cervenansky C, Marin M, Reiss C, Ehrlich R, Deana A (2002) Silent mutations affect in vivo protein folding in Escherichia coli. Biochem Biophys Res Commun 293(1):537–541CrossRefPubMedGoogle Scholar
  16. Cuccui J, Wren B (2015) Hijacking bacterial glycosylation for the production of glycoconjugates, from vaccines to humanised glycoproteins. J Pharm Pharmacol 67(3):338–350CrossRefPubMedGoogle Scholar
  17. de Groot NS, Castillo V, Grana-Montes R, Ventura S (2012) AGGRESCAN: method, application, and perspectives for drug design. Methods Mol Biol 819:199–220CrossRefPubMedGoogle Scholar
  18. Deana A, Ehrlich R, Reiss C (1998) Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo. Nucleic Acids Res 26(20):4778–4782CrossRefPubMedPubMedCentralGoogle Scholar
  19. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27(3):297–306CrossRefPubMedGoogle Scholar
  20. Denoncin K, Collet JF (2013) Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead. Antioxid Redox Signal 19(1):63–71CrossRefPubMedPubMedCentralGoogle Scholar
  21. Duong-Ly KC, Gabelli SB (2015) Affinity purification of a recombinant protein expressed as a fusion with the Maltose-Binding Protein (MBP) tag. Methods Enzymol 559:17–26CrossRefPubMedPubMedCentralGoogle Scholar
  22. Emtage JS, Angal S, Doel MT, Harris TJ, Jenkins B, Lilley G, Lowe PA (1983) Synthesis of calf prochymosin (prorennin) in Escherichia coli. Proc Natl Acad Sci U S A 80(12):3671–3675CrossRefPubMedPubMedCentralGoogle Scholar
  23. Fergus C, Barnes D, Alqasem MA, Kelly VP (2015) The queuine micronutrient: charting a course from microbe to man. Nutrients 7(4):2897–2929CrossRefPubMedPubMedCentralGoogle Scholar
  24. Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S (2011) Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J 278(14):2419–2427CrossRefPubMedGoogle Scholar
  25. Gavrilescu M, Chisti Y (2005) Biotechnology-a sustainable alternative for chemical industry. Biotechnol Adv 23(7–8):471–499CrossRefPubMedGoogle Scholar
  26. Georgiou G, Segatori L (2005) Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr Opin Biotechnol 16(5):538–545CrossRefPubMedGoogle Scholar
  27. Hammond B (2007) The food safety assessment of bovine somatotropin (bST). In: Hammond B (ed) Food safety of proteins in agricultural biotechnology. CRC Press, New York, pp 167–208. doi: 10.1201/9781420005738.ch7 CrossRefGoogle Scholar
  28. Hardin DS (2008) Treatment of short stature and growth hormone deficiency in children with somatotropin (rDNA origin). Biologics 2(4):655–661PubMedPubMedCentralGoogle Scholar
  29. Hayashi K, Kojima C (2008) pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production. Protein Expr Purif 62(1):120–127CrossRefPubMedGoogle Scholar
  30. Hess AK, Saffert P, Liebeton K, Ignatova Z (2015) Optimization of translation profiles enhances protein expression and solubility. PLoS One 10(5):e0127039CrossRefPubMedPubMedCentralGoogle Scholar
  31. Jackwood M, Hickle L, Kapil S, Silva R (2008) Vaccine development using recombinant DNA technology. Council Agric Sci Technol 38:1–11Google Scholar
  32. Kaplan W, Husler P, Klump H, Erhardt J, Sluis-Cremer N, Dirr H (1997) Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag. Protein Sci 6(2):399–406CrossRefPubMedPubMedCentralGoogle Scholar
  33. Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8(8):1668–1674CrossRefPubMedPubMedCentralGoogle Scholar
  34. Keefer LM, Piron MA, De Meyts P (1981) Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors. Proc Natl Acad Sci U S A 78(3):1391–1395CrossRefPubMedPubMedCentralGoogle Scholar
  35. Kroll J, Klinter S, Schneider C, Voss I, Steinbuchel A (2010) Plasmid addiction systems: perspectives and applications in biotechnology. Microb Biotechnol 3(6):634–657CrossRefPubMedPubMedCentralGoogle Scholar
  36. Kurland CG (1991) Codon bias and gene expression. FEBS Lett 285(2):165–169CrossRefPubMedGoogle Scholar
  37. Kuznetsova IM, Turoverov KK, Uversky VN (2014) What macromolecular crowding can do to a protein. Int J Mol Sci 15(12):23090–23140CrossRefPubMedPubMedCentralGoogle Scholar
  38. 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. Biotechnology 11(2):187–193CrossRefPubMedGoogle Scholar
  39. Leader B, Baca QJ, Golan DE (2008) Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov 7(1):21–39CrossRefPubMedGoogle Scholar
  40. Li Y (2011) Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 33(5):869–881CrossRefPubMedGoogle Scholar
  41. Lorsch JR (ed) (2015) Laboratory methods in enzymology: protein part D, vol 559. Meth Enzymol, 2015/06/23 edn, Academic Press (Elsevier)Google Scholar
  42. Lu Z, DiBlasio-Smith EA, Grant KL, Warne NW, LaVallie ER, Collins-Racie LA, Follettie MT, Williamson MJ, McCoy JM (1996) Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins. J Biol Chem 271(9):5059–5065CrossRefPubMedGoogle Scholar
  43. Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, Kohl TA, Corchero JL, Villaverde A, Schaffer L, Head SR, Souvignier C, Meredith TC, Woodard RW (2015) Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microb Cell Fact 14:57CrossRefPubMedPubMedCentralGoogle Scholar
  44. Miot M, Betton JM (2004) Protein quality control in the bacterial periplasm. Microb Cell Fact 3(1):4CrossRefPubMedPubMedCentralGoogle Scholar
  45. Neidhardt FC, Curtiss R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff W, Riley M, Schaechter M, Umbarger HE (1996) Escherichia coli and Salmonella: cellular and molecular biology, vol 1, 2nd edn. Society for Microbiology (ASM) Press, Washington, DCGoogle Scholar
  46. Olempska-Beer ZS, Merker RI, Ditto MD, DiNovi MJ (2006) Food-processing enzymes from recombinant microorganisms–a review. Regul Toxicol Pharmacol 45(2):144–158CrossRefPubMedGoogle Scholar
  47. Papaneophytou CP, Kontopidis G (2014) Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif 94:22–32CrossRefPubMedGoogle Scholar
  48. Peubez I, Chaudet N, Mignon C, Hild G, Husson S, Courtois V, De Luca K, Speck D, Sodoyer R (2010) Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microb Cell Fact 9:65CrossRefPubMedPubMedCentralGoogle Scholar
  49. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35 (Web Server issue):W126–131Google Scholar
  50. Rader R, Langer E (2015) Biopharmaceutical manufacturing: historical and future trends in titers, yields, and efficiency in commercial-scale bioprocessing. Bioprocess J 13(4):47–54CrossRefGoogle Scholar
  51. Ramon A, Senorale-Pose M, Marin M (2014) Inclusion bodies: not that bad. Front Microbiol 5:56CrossRefPubMedPubMedCentralGoogle Scholar
  52. Rosano GL, Ceccarelli EA (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Fact 8:41CrossRefPubMedPubMedCentralGoogle Scholar
  53. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172PubMedPubMedCentralGoogle Scholar
  54. Rudge P, Jaunmuktane Z, Adlard P, Bjurstrom N, Caine D, Lowe J, Norsworthy P, Hummerich H, Druyeh R, Wadsworth JD, Brandner S, Hyare H, Mead S, Collinge J (2015) Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years. Brain 138(Pt 11):3386–3399CrossRefPubMedPubMedCentralGoogle Scholar
  55. Ryan MP, Walsh G (2012) Veterinary-based biopharmaceuticals. Trends Biotechnol 30(12):615–620CrossRefPubMedGoogle Scholar
  56. Saez NJ, Vincentelli R (2014) High-throughput expression screening and purification of recombinant proteins in E. coli. Methods Mol Biol 1091:33–53CrossRefPubMedGoogle Scholar
  57. Salinas G, Pellizza L, Margenat M, Flo M, Fernandez C (2011) Tuned Escherichia coli as a host for the expression of disulfide-rich proteins. Biotechnol J 6(6):686–699CrossRefPubMedGoogle Scholar
  58. Sanchez-Garcia L, Martin L, Mangues R, Ferrer-Miralles N, Vazquez E, Villaverde A (2016) Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact 15(1):33CrossRefPubMedPubMedCentralGoogle Scholar
  59. Schafer F, Seip N, Maertens B, Block H, Kubicek J (2015) Purification of GST-tagged proteins. Meth Enzymol 559:127–139CrossRefPubMedGoogle Scholar
  60. Shur O, Dooley K, Blenner M, Baltimore M, Banta S (2013) A designed, phase changing RTX-based peptide for efficient bioseparations. Biotechniques 54(4):197–198, 200, 202, 204, 206Google Scholar
  61. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67(1):31–40CrossRefPubMedGoogle Scholar
  62. Spriestersbach A, Kubicek J, Schafer F, Block H, Maertens B (2015) Purification of His-tagged proteins. Meth Enzymol 559:1–15CrossRefPubMedGoogle Scholar
  63. Tyedmers J, Mogk A, Bukau B (2010) Cellular strategies for controlling protein aggregation. Nat Rev Mol Cell Biol 11(11):777–788CrossRefPubMedGoogle Scholar
  64. Valdez-Cruz NA, Caspeta L, Perez NO, Ramirez OT, Trujillo-Roldan MA (2010) Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb Cell Fact 9:18CrossRefPubMedPubMedCentralGoogle Scholar
  65. Vasina JA, Peterson MS, Baneyx F (1998) Scale-up and optimization of the low-temperature inducible cspA promoter system. Biotechnol Prog 14(5):714–721CrossRefPubMedGoogle Scholar
  66. Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298(5599):1790–1793CrossRefPubMedGoogle Scholar
  67. Walsh G (2014) Biopharmaceutical benchmarks 2014. Nat Biotechnol 32(10):992–1000CrossRefPubMedGoogle Scholar
  68. Yoon SH, Kim SK, Kim JF (2010) Secretory production of recombinant proteins in Escherichia coli. Recent Pat Biotechnol 4(1):23–29CrossRefPubMedGoogle Scholar
  69. Zalucki YM, Jones CE, Ng PS, Schulz BL, Jennings MP (2010) Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein. Biochim Biophys Acta 1798(6):1244–1249CrossRefPubMedGoogle Scholar
  70. Zalucki YM, Beacham IR, Jennings MP (2011) Coupling between codon usage, translation and protein export in Escherichia coli. Biotechnol J 6(6):660–667CrossRefPubMedGoogle Scholar
  71. Zambrano R, Jamroz M, Szczasiuk A, Pujols J, Kmiecik S, Ventura S (2015) AGGRESCAN3D (A3D): server for prediction of aggregation properties of protein structures. Nucleic Acids Res 43(W1):W306–W313CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Singapore 2016

Authors and Affiliations

  • Paula Tucci
    • 1
  • Victoria Veroli
    • 1
  • Mario Señorale
    • 1
  • Mónica Marín
    • 1
  1. 1.Biochemistry and Molecular Biology Section, Faculty of SciencesUniversidad de la RepúblicaMontevideoUruguay

Personalised recommendations