Inhibition of Aspergillus serine proteinase by Streptomyces subtilisin inhibitor and high-level expression of this inhibitor in Pichia pastoris.
Aspergillus fumigatus encodes an extracellular serine proteinase of the subtilisin family that is thought to be involved in invasive aspergillus infection of immunocompromised patients. When the structure of proteinase K was used to model this Aspergillus serine proteinase, Streptomyces subtilisin inhibitor (SSI) was predicted to be capable of binding to the serine proteinase. SSI purified from S. albogriseolus inhibited the serine proteinase with Ki of 1 x 10(-9)M. This value is higher than that for subtilisin probably because the serine proteinase lacks the S(4-6) site that interacts with the P(4-6) site of SSI. A high level expression for SSI, established in Pichia pastoris, yielded 0.5g of SSI per liter of culture medium. The secreted product was easily purified to homogeneity and biochemically characterized. The recombinant SSI showed a Ki of 1.1 x 10(-9) and 1 x 10(-8) for the serine proteinases from A. fumigatus and A. flavus, respectively.
Markaryan
A
A
Ohio State University, Neurobiotechnology Center, Columbus, 43210, USA.
Beall
C J
CJ
Kolattukudy
P E
PE
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D019286:Endopeptidase K; D015870:Gene Expression; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010843:Pichia / Q000235:genetics*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000494:pharmacology*
D001426:Bacterial Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D012697:Serine Endopeptidases; D019286:Endopeptidase K