Expression and characterization of serine proteinase inhibitor of the black tiger shrimp Penaeus monodon

Abstract

The five-domain Kazal-type proteinase inhibitor from the black tiger shrimp Penaeus monodon, SH415, was cloned and expressed in the Escherichia coli expression system. The 5' terminal truncated serine proteinase inhibitor gene containing 474 base pair open reading frame encoding for 248 amino acid polypeptide was cloned into pET-22b(+) between a pelB signal sequence and His Tag. The pET-226(+)-SPI clone was expressed in E. coli strain Rosetta (DE3) pLysS. The protein was expressed in the E. coli containing pET-22b(+)-SPI as the inclusion bodies and the identity of the expressed recombinant protein was confirmed by Western blot analysis. Two conditions were used to solubilize the inclusion bodies, the denaturing and non-denaturing or native conditions for further purification. The inhibitory activity of the recombinant protein, purified using denaturing condition was about 2 folds lower than that purified using native condition. The recombinant proteinprepared under the native condition was further purified using Sephadex G-100 chromatrography. It was purified up to 1.03 folds with 17% recovery. The molecular mass of recombinant SPI was determined by using MALDI-TOF Mass Spectrometry to be 29.065 kDa. From the proteinase inhibitory assay, the purified protein showed substantial inhibitory activity agianst subtilisin and elastase and low activity against trypsin. The inhibition constant (Ki) of subtilisin and elastase inhibitor complex were 0.26 and 3.16 nM, respectively.