Thesis (M.Sc.)--Chulalongkorn University, 2009

Serine proteinase inhibitors (SPIs) are found widely in multicellular organisms and function as regulators of proteinase activities involving in many biological processes. The four- and five-domain Kazal-type serine proteinase inhibitors, SPIPm1 and SPIPm2, were identified from the hemocyte cDNA library of the black tiger shrimp, Peneaus monodon. From the previous study, The SPIPm2 was found to strongly inhibited subtilisin and elastase, and weakly inhibited trypsin. In this study, the SPIPm2 gene was mutated by site-directed mutagenesis in order to delineate the domain inhibitory activities. The P1 amino acid residues of domains 1, 2 and 3 were changed from Thr to Leu (T35L), Ala to Trp (A83W) and Glu to Leu (E131L), respectively. The wild type and three mutated SPIPm2 proteins were produced and their inhibitory specificities against proteinases were determined. We found that T35L and E131L slighty inhibited trypsin less than the wild type. Only A83W had no elastase inhibition activity. All mutant proteins had no inhibitory activity against chymotrypsin like the wild type. The individual mutated domain 3, D3E131L, was produced to study the effect of P1 residue mutation on the inhibitory specificity. The D3E131L had the strong inhibitory activity against subtilisin and elastase. Moreover, the SPIPm1, which have 669 bp of open reading frame coding for 222 amino acids, was cloned in to pET-28b(+) and expressed by using E. coli system. The protein was expressed as the inclusion bodies and the identity of the expressed recombinant protein was confirmed by Western blot analysis. The denaturing condition was used to solubilize the inclusion bodies and the solubilized protein was further purified using nickel-NTA column. From the proteinase inhibitory assay, the SPIPm1 showed the weak inhibition against all proteinases when the mole ratio of inhibitor against proteinases was 80 : 1.