牛胰磷脂? A2 之定位變造： 22 , 113 , 115 , 118 等位置與活性及神經毒性的相關性

Abstract

先設計一條帶有擬突變位置的引子，再以具有插入牛胰磷脂?A2基因的質體pTO–A2M為範本，以PCR的方式得到帶有欲突變位置的PLA2基因。再將此DNA片段接合到表現載體pTO-A2M上，使其轉化E.coli DE3菌種，然後以IPTG誘導突變的PLA2蛋白質之生合成。在適當的反應條件下重新纏繞，而形成正確的結構。再經Poros Hs管柱及C-18 Reverse Phase HPLC純化後收集正確纏繞的PLA2蛋白質。 以此方法製得五個變異的PLA2蛋白質，分別為F22W(22位置胺基酸由Phe變為Trp)；K113Y, H115Y, L118Y, H115Y+L118Y(113位置的Lys，115位置的His，118位置的Leu變為Tyr及將115位置的His與118位置的Leu一起變為Tyr）。分別測定這些突變的磷脂?A2酵素活性及對125I-Taipoxin與神經膜結合之抑制。所得的結果顯示F22W位置突變的磷脂?A2喪失了酵素活性，而其他位置的突變分別失去15?55％的酵素活性。在以125I-Taipoxin作Equilibrium Binding Assay和Cross-linking experiment所得的結果顯示，所有突變的磷脂?A2皆不會與125I-Taipoxin競性對神經鍵體的膜蛋白（Synaptic membrane protein）之結合。因此推論這些突變位置與酵素活性有某種程度的相關性，而與神經毒的特性沒有相關性。

Site-directed mutagenesis was used to generate mutants of bovine pancreatic phospholipase A2 for studying the structure and functions of the protein. The pTO-A2M plasmid, which bovine pancreatic PLA2 gene has been inserted into, was used as template and designed oligonucleotide fragments as primers in polymerase chain reaction to obtain cDNA for PLA2 with desired mutation sites. The DNA was inserted into the expression vector, pTO-A2M, and the plasmid was transfected into the E.coli DE3 strain. The expressed PLA2 mutant proteins were extracted, correctly refolded and then purified by ion exchange chromatography and reversed phase HPLC. In this way, five mutant proteins were obtained. They are F22W, K113Y, H115Y, L118Y and H115Y+L118Y. The enzyme activity of each mutant protein was assayed by the pH-stat method using egg lecithin as substrate. The mutant protein F22W completely lacks PLA2 enzyme activity, and the other mutant proteins show little change in the enzyme activity. All five mutants are unable to compete in both equilibrium binding assay and cross-linking experiment with 125I-Taipoxin for binding to synaptic membrane proteins.