Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli.
Rat liver protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds. We have developed an efficient method for its overproduction in Escherichia coli. Using a T7 RNA polymerase expression system, isolated yields of 15-30 mg/liter of recombinant rat PDI are readily obtained. Convenient purification of the enzyme from E. coli lysates involves ion-exchange (DEAE) chromatography combined with zinc chelate chromatography. The recombinant PDI shows catalytic activity identical to that of PDI isolated from bovine liver in both the reduction of insulin and the oxidative folding of ribonuclease A. The enzyme is expressed in E. coli as a soluble, cytoplasmic protein. After complete reduction and denaturation in 6 M guanidinium hydrochloride, PDI regains complete activity within 3 min after removal of the denaturant, implying that disulfide bonds are not essential for the maintenance of PDI tertiary structure. Both the protein isolated from E. coli and the protein isolated from liver contained free cysteine residues (1.8 +/- 0.2 and 1.4 +/- 0.3 SH/monomer, respectively).
Gilbert
H F
HF
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Texas 77030.
Kruzel
M L
ML
Lyles
M M
MM
Harper
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D011994:Recombinant Proteins; D004247:DNA; D007535:Isomerases; D019704:Protein Disulfide-Isomerases