title: New vectors for high level expression of recombinant proteins in bacteria.
abstract: A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
authors.forename: D J
authors.initials: DJ
authors.lastname: Hakes
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000502:physiology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D013917:Thrombin / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D013917:Thrombin