2A型蛋白去磷酸化酵素催化體在大腸桿菌中的高度表達   全文下載

Overexpression of the Catalytic Subunit of Protein Phosphatase Type 2A in Escherichia coli

本研究的目的係利用分子選種方式，發展一套能在大腸桿菌中高度表達牛腎上腺髓質2A型蛋白去磷酸化酵素催化體的系統。大量的酵素經分離，純化後，將可供進一步研究該2A型酵素的結構及其活性基位和其催化體與調節體之相互關係。2A型酵素催化體已成功的在大腸桿菌中獲得表達，所使用的質體pQE31含有大腸桿菌噬菌體T5驅動子（T5 promoter）和兩段Iac operator作為2A型酵素的表達和調控，另一段合成6個histidine標的胜?基因以便利所表達的2A型酵素之純化和分離。所表達出來之酵素約佔菌體蛋白的30～35%，但大都為水不溶性蛋白且不具酵素活性。SDS-PAGE分析顯示，其分子量為36kDa，與自人的紅血球分離到之2A型酵素相同。且此基因重組蛋白和紅血球分離出來的2A型酵素一樣，能與合成2A型酵素胜?（相對於2A型酵素催化體氨基酸順序296-309）之特異抗體作用。此不溶性蛋白可溶解於6M guanidine hydrochloride，並直接利用Ni-NTA resin來純化，單一的吸附層析純化可達90%以上的純化率，且完全的純化可再藉由通過凝膠過濾層析來達成。大約每公升的培養基可獲得15～20mg純化之2A型酵素。目前，酵素回性條件尚在探討中，若能有效的使蛋白再摺疊而活化該酵素，則本表達系統提供一個大量生產且快速純化2A型酵素的方法，將有助於探討該酵素的結構和其生理的功能。

In this study we have established a system for expression of the catalytic subunit of protein phosphatase type 2A (PP2A) of bovine adrenal medulla in E. coli. Expression of PP2A in pQE31 vector, based on use of the phage T5 promoter and two lac operator sequences, produced the enzyme as an insoluble aggregate which constituted up to 30-35% of total cellular protein. Although enzyme activity was not detected, the protein had an Mr of 36 kDa, comparable to that of authentic phosphatase 2A isolated from human red blood cells. Both the recombinant protein and the authentic phosphatase 2A exhibited cross-reactivity with specific antibody against a synthetic peptide corresponding to PP2A amino acid residues 296-309. The insoluble protein, fused with a 6x His affinity tag was directly applied to a Ni-NT A column for purification by affinity chromatography. Approximately 90% homogeneity was obtained by a single purification and the enzyme was essentially homogeneous after further passage through a gel filtration column. These procedures yielded about 15-20 mg purified PP2A/liter culture, which will facilitate further renaturation experiments and future studies on structure-function relationships of the enzyme.