Thesis (M.Sc.)--Chulalongkorn University, 2003

Snake venom phospholipaseA[subscript 2] (PLA[subscript 2]) enzymes are low molecular weight (13-15 kDa) proteins and exhibit a wide variety of pharmacological effects such as hemolysis, platelet aggregation inhibition, neurotoxicity and myonecrosis. Russells viper (Daboia russellii siamensis, RV) venom contains several isoforms of PLA2 different from each subspecies. The aim of this study was to search for the PLA[subscript 2] isoforms in a Thai Daboia russellii siamensis venom gland cDNA library, to develop a method of recombinant expression of PLA[subscript 2] by genetic engineering and study PLA[subscript 2]s gene structure. By using plaque-lift hybridization and random selection of cDNA library clone for DNA sequence determination, we found only 2 PLA[subscript 2] isoforms in the Thai RV venom gland cDNA library with ratio approximately 1:2. The 2 PLA[subscript 2] isoforms, designated PlaS1 and PlaS2, showed 86% nucleotide sequence identity and are identical to Taiwan Russell's viper PLA2, RV-4 and RV-7, respectively. The PLA[subscript 2] isoforms were recombinantly expressed as a histidine tagged fusion protein in using an E. coli and yielded 18 kDa and 23 kDa proteins. Because of low level of expression, only the 18 kDa PlaS1 was expressed in a large scale, purified by using Immobilized Metal Affinity Chromatography (IMAC) under denaturing condition, solubilized, refolded by dialysis and showed PLA[subscript 2] activity of 185.67 mmoles/min/mg. The yield of recombinant protein was about 2.0 microgram/Litre of cells culture. We cloned the full-length genes encoding PLA2 by PCR of genomic DNA from muscle tissue of an RV. We identified 3 genes about 2.0 kb in length, designated gPlaS1, gPlaS2 and gPlaS3, including 5 exons and 4 introns in genomic PLA[subscript 2] DNA cloning. The coding and untranslated regions of gPlaS1 and gPlaS2 are identical to PlaS1 and PlaS2 cDNA, respectively. The deduced N-terminal amino acid sequences of gPlaS3, however, is different from all previous reported of D. russellii PLA[subscript 2]s.