Introduction to Expression by Fusion Protein Vectors

Paul Riggs1, Edward R. La Vallie2, John M. McCoy2

1 New England Biolabs, Beverly, Massachusetts, 2 Genetics Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.4A
DOI:  10.1002/0471142727.mb1604as28
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This overview discusses issues involved with creating and manipulating vectors for expression of fusion proteins. The requirements for efficient translation include a promoter and a start codon, along with the fact that the mRNA encoding the protein to be expressed must contain a ribosome‐binding site that is not blocked by mRNA secondary structure. The level of expression is also affected by codon preferences, and may be affected by the coding sequence in other ways that are not yet well understood. In virtually all cases, these problems can be solved by altering the sequence preceding the start codon, and/or by making changes in the 5′ end of the coding sequence that do not change the protein sequence, taking advantage of the degeneracy of the genetic code. However, it is often quicker to solve these problems by making fusions between genes. In this approach the cloned gene is introduced into an expression vector 3′ to a sequence (carrier sequence) coding for the amino terminus of a highly expressed protein (carrier protein). The carrier sequence provides the necessary signals for good expression, and the expressed fusion protein contains an N‐terminal region encoded by the carrier. The carrier sequence can also code for an entire functional moiety or even for an entire protein that can be exploited in purifying the protein, either with antibodies or with an affinity purification specific for that carrier protein. Alternatively unique physical properties of the carrier protein (e.g., heat stability) can be exploited to allow selective purification of the fusion protein. Often, proteins fused to these carriers can be separated from the bulk of intracellular contaminants by taking advantage of special attributes.

PDF or HTML at Wiley Online Library

Table of Contents

  • Solubility of the Expressed Protein
  • Stability of the Expressed Protein
  • Cleavage of Fusion Proteins to Remove the Carrier
  • Literature Cited
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Amann, E. and Brosius, J. 1985. “ATG vectors” for regulated high‐level expression of cloned genes in Escherichia coli. Gene 40:183‐190.
   Baker, T.A., Grossman, D., and Gross, C.A. 1984. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc. Natl. Acad. Sci. U.S.A. 81:6779‐6783.
   Bishia, 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.
   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.
   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 proteolysis. Proc. Natl. Acad. Sci. U.S.A. 81:4692‐4696.
   Grodberg, J. and Dunn, J.J. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 170:1245‐1253.
   Haffey, M.L., Lehman, D., and Boger, J. 1987. Site‐specific cleavage of a fusion protein by renin. DNA 6:565‐571.
   Itakura, K., Hirose, T., Crea, R.M., Riggs, A.D., Heyneker, H.L., Bolivar, F., and Boyer, H.W. 1977. Expression in E. coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1053‐1056.
   LaVallie, E.R., Rehemtulla, A., Racie, L.A., DiBlasio, E.A., Ferenz, C., Grant, K.L., Light, A., and McCoy, J.M. 1993. Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268:23311‐23317.
   Lee, N., Cozzikorto, J., Wainwright, N., and Testa, D. 1984. Cloning with tandem gene systems for high level gene expression. Nucl. Acids Res. 12:6797‐6812.
   Nagai, K. and Thøgersen, H. C. 1984. Generation of β‐globin by sequence‐specific proteolysis of a hybrid protein produced 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.
   Schein, C.H. 1989. Production of soluble recombinant proteins in bacteria. Bio/Technology. 7:1141‐1149.
   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.
   Stormo, G.D., Schneider, T.D., and Gold, L. 1982. Characterization of translation initiation sites in E. coli. Nucl. Acids Res. 10:2971‐2996.
   Strauch, K.L. and Beckwith, J. 1988. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc. Natl. Acad. Sci. U.S.A. 85:1576‐1580.
   Sugimura, K. and Higashi, N. 1988. A novel outer‐membrane‐associated protease in Escherichia coli. J. Bacteriol. 170:3650‐3654.
   Szoka, P.R., Schreiber, A.B., Chan, H., and Murthy, J. 1986. A general method for retrieving the components of a genetically engineered fusion protein. DNA 5:11‐20.
PDF or HTML at Wiley Online Library