Purification And Expression Of Soluble Gln49-PLA₂ From Agkistrodon Blomhoffii Snake Venom And Its Site-directed Mutagenesis

A novel basic phospholipase A₂, named Gln49-PLA₂, was purified from Agkistrodon blomhoffii ussurensis snake venom. Its molecular mass is 13880.798 kDa assayed by mass spectrometry and isoelectric point is 8.56 analyzed by isoelectric focusing electrophoresis. Myotoxic, cytotoxic and the activity of thrombin-like serine protease had been detected, except for the activity of hydrolysis of phospholipids. Gln49-PLA₂ contains 14 cysteine (Cys) forming 7 disulfide bonds, which is a good model to study the relationship between structures and effects. Therefore, the gene of Gln49-PLA₂ and genes of its mutagenesis, which were constructed in vector pET-32a (+) with PCR site-directed mutagenesis and Gene Cloning, expressed as inclusion body in E.coli, but the results were not ideal. The main purpose of this dissertation is to make an attempt to get an efficient soluble expression of fusion Gln49-PLA₂ in vector pMAL c2x through Gene Cloning.Regene Gln49-PLA₂ and its mutant Lys49-PLA₂ and Asp49-PLA₂ etc. were obtained by overlap extention PCR. After double digested with AvaI and BamHI, ligated of Gln49-PLA₂ and its mutant gene fragment to pMAL with T4 ligase, purified with Qiagen spin column and transformed to ER2984, white colony was screened. The target recombinant plasmids, minipreped by Qiagen spin column, were identified by double digestion, agarose gel electrophoresis assay and DNA sequencing. The target recombinant plasmid was transformed into E.coli TB1. SDS-PAGE result of induced cells with fusion proteins certified its soluble expression. Optimized expression conditions of fmAsp49-PLA₂, an efficient soluble expression of fmAsp49-PLA₂ were obtained. The fmAsp49-PLA₂ was purified from the bacterial periplasm by affinity chromatography in an amylose column and by anion exchange chromatography. Attempts to cleave the fmAsp49-PLA₂ selectively at the factor Xa recognition site was successful, SDS-PAGE results demonstrated the completement of cleavage, in which one fragment of approximate 40 kDa and the other of approximate 14 kDa were detected, with the optimized conditions including cleavage temperature 4℃, protein concentration 0.8 mg/ml, cleavage time 12h. By three steps method, HiTrap SP cation exchange, HiTrap Heparin and Superdex 75 gel-filtration, we failed to get pure mAsp49-PLA₂ from fmAsp49-PLA₂. PLA₂ activity and coagulation activity of the fmAsp49-PLA₂ and fGln-PLA₂ were detected, both of them were low.