Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants.
A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His(6)-PSP was 1.5 mg/g biomass.
Khairullin
R F
RF
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Mikhailova
A G
AG
Sebyakina
T Yu
TY
Lubenets
N L
NL
Ziganshin
R H
RH
Demidyuk
I V
IV
Gromova
T Yu
TY
Kostrov
S V
SV
Rumsh
L D
LD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012705:Serratia / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014361:Trypsin Inhibitors / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases; C090703:oligopeptidase B