Production of human protein disulfide isomerase by Bacillus brevis.
Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B. brevis as vector. The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l-1 in the late exponential phase of the bacterial growth. The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence. It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds. The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione. These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.
Tojo
H
H
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Asano
T
T
Kato
K
K
Udaka
S
S
Horiuchi
R
R
Kakinuma
A
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D019704:Protein Disulfide-Isomerases; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases