A procedure for the generation and the purification of Escherichia coli thioredoxins with variable N-terminal sequences.
We have developed a rapid and simple procedure for the production and the purification of Escherichia coli thioredoxins containing additional amino acid residues at the N-terminus. By the polymerase chain reaction, the complete gene encoding for E. coli thioredoxin was modified and amplified with the addition at its 5' end of a BamHI cloning site and a triplet coding for an arginine residue instead of the initiator methionine codon, whereas at the 3' end the stop codon was followed by an EcoRI cloning site. The synthetic DNA was ligated into the BamHI/EcoRI site of the vector plasmid pGEX-2T, and the novel plasmid [pFTG] was used for the transformation of E. coli cells. Following induction and cell disruption, a protein composed of Schistosoma japonicum glutathione S-transferase and E. coli thioredoxin was obtained in soluble form and purified by affinity chromatography on agarose columns bearing immobilized glutathione. This procedure yielded 50 mg of homogeneous fusion protein per liter of culture media. Digestion of the chimeric thioredoxin with bovine plasma thrombin followed by an additional chromatography on glutathione-agarose gave a protein that contained the entire sequence of E. coli thioredoxin and three additional amino acid residues [G-S-R-] at the N-terminal side. The structural characteristics and the protein disulfide oxidoreductase activity of this recombinant protein, in terms of variations of emission fluorescence and reduction of insulin disulfide bonds, respectively, were essentially identical to those of its counterpart obtained from wild-type cells by conventional techniques of proteins purification.(ABSTRACT TRUNCATED AT 250 WORDS)
Mora-García
S
S
Instituto de Investigaciones Bioquímicas (Fundación Campomar, F.C.E.N.-U.B.A., CONICET), Argentina.
Hagelin
K
K
Wolosiuk
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification*
D007328:Insulin; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D005982:Glutathione Transferase