Recombinant expression and characterization of five-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus monodon)

Abstract

Four full-length complementary DNAs of Kazal-Type serine proteinase inhibitors (SPIs) were identified from hemocyte cDNA libraries of the black tiger shrimp Penaeus monodon and recognized as having 4 (SPIPm1) and 5 (SPIPm2, SPIPm3, and SPIPm4) Kazal domains. SPIPm2 encoding a complete 5-Kazal-domain inhibitor was expressed in the Escherichia coli system. Inhibitory activity of crude proteins against various serine proteases was tested using SPI activity gelatin with sodium dodecylsulfate polyacrylamide gel electrophoresis and inhibitory spectrum assays. A 32-kDa recombinant protein (rSPIPm2) showed inhibitory activity against trypsin, chymotrypsin, and subtilisin, but not elastase. Concordantly, inhibitory spectrum assays showed that crude rSPIPm2 strongly inhibited trypsin (89%) and chymotrypsin (70%), but less effectively inhibited subtilisin (8%), and did not inhibit elastase activity. Northern blot analysis of hemocyte total RNA showed 2 SPI transcripts of 1.6 and 1.7 kb in size. Tissue-specific expression using reverse transcriptase polymerase chain reaction suggests that SPIPm2 is exclusively expressed in hemocytes of P. monodon.