Recombinant Expression and Characterization of Fiv

Authors: Boonyarin Jarasrassamee Premruethai Supungul Sakol Panyim Sirawut Klinbunga Vichien Rimphanichayakit Anchalee Tassanakajon

Publish Date: 2005/03/11

Volume: 7, Issue: 1, Pages: 46-52

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Abstract

Four fulllength complementary DNAs of KazalType 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 5Kazaldomain 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 32kDa 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 16 and 17 kb in size Tissuespecific expression using reverse transcriptase polymerase chain reaction suggests that SPIPm2 is exclusively expressed in hemocytes of P monodonThis work was supported by a research grant BTB062B09309 from the National Center for Genetic Engineering and Biotechnology BIOTEC to A Tassanakajon and by the Thailand Research Fund’s Senior Research Scholar award to S Panyim The Thailand Graduate Institute of Science and Technology TGIST Student Fellowship to B Jarasrassamee and BIOTEC Postdoctoral Fellowship to P Supungul are also acknowledged

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