Production of Recombinant Proteins in Escherichia coli

Edward R. LaVallie1

1 Genetics Institute, Inc., Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.1
DOI:  10.1002/0471140864.ps0501s00
Online Posting Date:  May, 2001
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E. coli is the expression system of choice and a substantial body of literature has accumulated on the successful expression of foreign genes in this host. Several problems with protein expression in E. coli have been encountered, and many have been ultimately solved. This unit describes methods that have been developed for production of recombinant proteins in E. coli and potential pitfalls that may be encountered.

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Table of Contents

  • General Strategies for Gene Expression in E. Coli
  • Specific Expression Strategies
  • Summary
  • Literature Cited
  • Tables
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Literature Cited

Literature Cited
   Bishai, W.R., Rappuoli, R., and Murphy, J.R. 1987. High‐level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. J. Bacteriol. 169:5140‐5151.
   Bornstein, P. and Balian, G. 1977. Cleavage at Asn‐Gly bonds with hydroxylamine. Methods Enzymol. 47:132‐145.
   Bowden, G.A. and Georgiou, G. 1990. Folding and aggregation of β‐lactamase in the periplasmic space of Escherichia coli. J. Biol. Chem. 265:16760‐16766.
   Brewer, S.J. and Sassenfeld, H.M. 1985. The purification of recombinant proteins using C‐terminal poly‐arginine fusions. Trends Biotechnol. 3:119‐122.
   Bucheler, U.S., Werner, D., and Schirmer, R.H. 1990. Random silent mutagenesis in the initial triplets of the coding region: A technique for adapting human glutathione eductase‐encoding cDNA to expression in Escherichia coli. Gene 96:271‐276.
   Cheah, K.C., Harrison, S., King, R., Crocker, L., Well, J.R., and Robins, A. 1994. Secretion of eukaryotic growth hormones in Escherichia coli is influenced by the sequence of the mature proteins. Gene 138:9‐15.
   Dalbøge, H., Dahl, H.H.M., Pedersen, J., Hansen, J.W., and Christensen, T. 1987. A novel enzymatic method for production of authentic hGH from an Escherichia coli‐produced hGH precursor. Bio/Technology 5:161‐164.
   De Lamarter, J.F., Mermod, J.J., Liang, C.M., Eliason, J.F., and Thatcher, D.R. 1985. Recombinant murine GM‐CSF from E. coli has biological activity and is neutralized by a specific antiserum. EMBO J. 4:2575‐2581.
   Devlin, P.E., Drummond, R.J., Toy, P., Mark, D.F., Watt, K.W.K. and Devlin, J.J. 1988. Alteration of the amino‐terminal codons of human granulocyte colony‐stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in Escherichia coli. Gene 65:13‐22.
   Dykes, C.W., Bookless, A.B., Coomber, B.A., Noble, S.A., Humber, D.C., and Hobden, A.N. 1988. Expression of atrial natriuretic factor as a cleavable fusion protein with chloramphenicol acetyltransferase in Escherichia coli. Eur. J. Biochem. 174:411‐416.
   Fuh, G., Mulkerrin, M.G., Bass, S., McFarland, N., Brochier, M., Bourell, J.H., Light, D.R., and Wells, J.A. 1990. The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. J. Biol. Chem. 265:3111‐3115.
   Gardella, T.J., Rubin, D., Abou‐Samra, A.‐B., Keutmann, H.T., Potts, J.T. Jr., Kronenberg, H.M., and Nussbaum, S.R. 1990. Expression of human parathyroid hormone (1‐84) in Escherichia coli as a factor X‐cleavable fusion protein. J. Biol.Chem. 265:15854‐15859.
   Gearing, D.P., Nicola, N.A., Metcalf, D., Foote, S., Willson, T.A., Gough, N.M. and Williams, R.L. 1989. Production of leukemia factor in Escherichia coli by a novel procedure and its use in maintaining embryonic stem cells in culture. Bio/Technology 7:1157‐1161.
   Germino, J. and Bastia, D. 1984. Rapid purification of a gene product by genetic fusion and site specific roteolysis. Proc. Natl. Acad. Sci. U.S.A. 81:4692‐4696.
   Guan, C., Li, P., Riggs, P.D., and Inouye, H. 1988. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose‐binding protein. Gene 67:21‐30.
   Haffey, M.L., Lehman, D., and Boger, J. 1987. Site‐specific cleavage of a fusion protein by renin. DNA 6:565‐571.
   Hellman, J. and Mantsala, P. 1992. Construction of an E. coli export‐affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin lucanotransferase. J. Biotechnol. 23:19‐34.
   Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F., and Boyer, H.W. 1997. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056‐1063.
   Knott, J.A., Sullivan, C.A., and Weston, A. 1988. The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli. Eur. J. Biochem. 174:405‐410.
   LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., and McCoy, J.M. 1993a. A thioredoxin gene fusion system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11:187‐193.
   LaVallie, E.R., Rehemtulla, A., Racie, L.A., DiBlasio, E.A., Ferenz, C., Grant, K.L., Light, A., and McCoy, J.M. 1993b. Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268:23311‐23317.
   Looman, A.C., Bodlaender, J., Comstock, L.J., Eaton, D., Jhurani, P., deBoer, H.A., and van Knippenberg, P.H. 1987. Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 6:2489‐2492.
   Lundeberg, J., Wahlberg, J., and Uhlen, M. 1990. Affinity purification of specific DNA fragments using a lac repressor fusion protein. Genet. Anal. Techn. Appl. 7:47‐52.
   Lunn, C.A., Kathju, S., Wallace, B.J., Kushner, S.R., and Pigiet, V. 1984. Amplification and purification of plasmid‐encoded thioredoxin from Escherichia coli K12. J. Biol. Chem. 259:10469‐10474.
   Maina, C.V., Riggs, P.D., Grandea, A.G., Slatko, B.E., Moran, L.S., Tagliamonte, J.A., McReynolds, L.A., and Guan, C. 1988. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose‐binding protein. Gene 74:365‐373.
   Mieschendahl, M., Petri, T., and Hanggi, U. 1986. A novel prophage independent trp regulated lambda pL expression system. Bio/Technology 4:802‐808.
  Miller, J.H. and Reznikoff, W.S., (eds.) 1978. The Operon. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Nagai, K. and Thøgersen, H.C. 1984. Generation of β‐globin by sequence‐specific proteolysis of a hybrid protein in Escherichia coli. Nature 309:810‐812.
   Nagai, K. and Thøgersen, H.C. 1987. Synthesis and sequence‐specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 153:461‐481.
   Neu, H.C. and Heppel, L.A. 1965. Release of enzymes rom Escherichia coli by osmotic shock during the formation of spheroplasts. J. Biol. Chem. 240:3685‐3692.
   Nilsson, B., Abrahmsen, L., and Uhlen, M. 1985. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4:1075‐1080.
   Oka, T., Sakamoto, S., Miyoshi, K., Fuwa, T., Yoda, K., Yamasaki, M., Tamura, G., and Miyake, T. 1985. Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82:7212‐7216.
   Olins, P.O. and Rangwala, S.H. 1989. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J. Biol. Chem. 264:16973‐16976.
   Persson, M., Bergstrand, M.G., Bulow, L., and Mosbach, K. 1988. Enzyme purification by genetically attached polycysteine and polyphenylalanine tags. Anal. Biochem. 172:330‐337.
   Power, B.E., Ivancic, N., Harley, V.R., Webster, R.G., Kortt, A.A., Irving, R.A., and Hudson, P.J. 1992. High‐level temperature‐induced synthesis of an antibody VH‐domain in Escherichia coli using the PelB secretion signal. Gene 113:95‐99.
   Racie, L.A., McColgan, J.M., Grant, K.L., DiBlasio‐Smith, E.A., McCoy, J.M., and LaVallie, E.R. 1995. Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA. Bio/Technology. In press.
   Robinson, M., Lilley, R., Little, S., Emtage, J.S., Yarranton, G., Stephens, P., Millican, A., Eaton, M., and Humphreys, G. 1984. Codon usage can affect efficiency of translation of genes in Escherichia coli. Nucl. Acids Res. 12:6663‐6671.
   Ruther, U. and Muller‐Hill, B. 1983. Easy identification of cDNA clones. EMBO J. 2:1791‐1794.
   Schatz, P.J. 1993. Use of peptide libraries to map the substrate specificity of a peptide‐modifying enzyme: A 13 residue consensus peptide specifies biotinylation in Escherichia coli. Bio/Technology 11:1138‐1143.
   Schein, C.H. 1989. Production of soluble recombinant proteins in bacteria. Bio/Technology 7:1141‐1149.
   Schein, C.S. and Noteborn, M.H.M. 1988. Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6:291‐294.
   Shimatake, H. and Rosenberg, M. 1981. Purified λ regulatory protein cII positively activates promoters for lysogenic development. Nature 292:128‐132.
   Shine, J. and Dalgarno, L. 1974. The 3′‐terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. U.S.A. 71:1342‐1346.
   Skerra, A. 1994. A general vector, pASK84, for cloning, bacterial production, and single‐step purification of antibody Fab fragments. Gene 141:79‐84.
   Smith, D.B. and Johnson, K.S. 1988. Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S‐transferase. Gene 67:31‐40.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60‐89.
   Szoka, P.R., Schreiber, A.B., Chan, H., and Murthy, J. 1986. A general method for retrieving components of a genetically engineered fusion protein. DNA 5:11‐20.
   Takahara, M., Sagai, H., Inouye, S., and Inouye, M. 1988. Secretion of human superoxide dismutase in Escherichia coli. Bio/Technology 6:195‐198.
   Taylor, M.E. and Drickamer, K. 1991. Carbohydrate‐recognition domains as tools for rapid purification of recombinant eukaryotic proteins. Biochem. J. 274:575‐580.
   Vasquez, J.R., Evnin, L.B., Higaki, J.N., and Craik, C.S. 1989. An expression system for trypsin. J. Cell. Biochem. 39:265‐276.
   Villa, S., DeFazio, G., and Canosi, U. 1989. Cyanogen bromide cleavage at methionine residues of polypeptides containing disulfide bonds. Anal. Biochem. 177:161‐164.
   Yansura, D.G. 1990. Expression as trpE fusion. Methods Enzymol. 165:161‐166.
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