Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.
Guan
K L
KL
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Dixon
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D017027:Protein Tyrosine Phosphatases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013917:Thrombin / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D013917:Thrombin; D005998:Glycine