| Phospholipase A2is widely exisited in nature with diverse biological activities such as anticancer, antimicrobial activity, anticoagulation and antithrombosis, which has the potency to be a resource for developing new pharmaceuticals. Moreover, PLA2can be used as an excellent model in the study of the relationship between protein structure and function because of its unique structure.A new toxin was isolated from Gloydius ussurensis snake venom in our lab before. The toxin was characterized as phosphlipase A2homologue with Gin at position49, named Gln49-PLA2. Gln49-PLA2exhibited activities of neurotoxicity, myotoxicity and thrombin-like serine protease, whereas phospholipase hydrolytic activity was not detected. Amino acid sequence alignment and phylogenetic tree analysis indicate that Gln49-PLA2was sequencely and genetically closer to Asp49-PLA2(93%to61%) than Lys49-PLA2(60%to56%). Further bioinformatics analysis and tertiary structure simulation showed that amino acid residue glutamine at49site might disturb its binding with Ca2+when compared to Asp49-PLA2from Gloydius halys snake venom, and this probablely would be the key factor for the loss of Gln49-PLA2;s phospholipase hydrolytic activity requiring Ca2+. As a result, a site-directed mutagenesis of Gin to Asp at position49on Gln49-PLA2had been carried out in purpose of confirming the role of amino acid residue Gln49in its activity of phospholipase hydrolysis. Gln49-PLA2and its mutants were expressed as inclusion body in E. coli, which was adverse to the research of protein structure and function. This paper aims to develop a soluble expression system of Gln49-PLA2and its mutant genes.In this study, we expressed Gln49-PLA2and its mutant Asp49-PLA2in the baculovirus expression system. Genes of Gln49-PLA2or Asp49-PLA2and EGFP gene were sequentially and respectively inserted into the downstream multiple cloning site of promoter PH and P10on the donor plasmid pFastBacTM Dual. Recombinant plasmid harboring the above genes was transformed into E. coli DHlOBac. Target genes were integrated into the genome of baculovirus by transposition, and a recombinant shuttle vector Bacmid-EGFP-Gln49/Asp49-PLA2was constructed. The recombinant bacmid was used to transfect Sf9cells mediated with lipofectin. Then the recombinant baculovirus was obtained, which subsequently infected Sf9cells to express Gln49-PLA2or Asp49-PLA2. SDS-PAGE analysis showed a soluble expression of Gln49-PLA2and Asp49-PLA2in the extracted supernatant from the cell lysate with molecular weight about14kDa. Recombinant proteins were purified by Hitrap SP cation exchange column. All purified proteins were characterized by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and experimental molecular masses of Gln49-PLA2and Asp49-PLA2suggested their successful expression in the baculovirus system. The purified Gln49-PLA2protein showed a hydrolysis activity of fibrinogen as natural source Gln49-PLA2and phospholipase hydrolytic activity was not detected. However, the mutant Asp49-PLA2exhibited a specific enzymatic activity of～3.33U/μg for hydrolyzing soybean lecithin substrate and a hydrolysis activity of fibrinogen was not detected, which approved the important role of Gln49and that the character of amino acid residue at position49might primarily determined the activity of Gln49-PLA2, as evidenced by the substitution of Gln49to Asp49. However, recombinant expression of Gln49-PLA2and Asp49-PLA2in baculovirus system is time-consuming and proteins degradation occurred in the process of purification. In order to avoid the degradation of target proteins by protease cleavage, cell-free protein synthesis system for Asp49-PLA2was attempted. SDS-PAGE showed a protein band of14kDa and the purified Asp49-PLA2exhibited the activity of phospholipase hydrolysis. Altogether, the results preliminarily indicated a successful expression of Asp49-PLA2in the E. coli cell-free protein synthesis system. |
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