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Study on the Renaturation of Mutant Lysozymes Expressed in Schericia Coli.

大肠杆菌中表达的突变型溶菌酶的复性研究。

基本信息

批准号：
01571214
负责人：
YAMADA Hidenori
金额：
$ 1.34万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Expression of foreign proteins having disulfide bonds in schericia coli usually results in the formation of insoluble and denatured proteins called inclusion body. This greatly reduces the advantage of the expression system in E. coli. In order to improve the utility of the expression system in E. coli. In order to improve the utility of the expression system in E. coli, we investigated the solubilization, purity, processing of the N-terminal methionine residue, and renaturation of mutant chicken lysozymes expressod in E. coli, and the following results were obtained.1. Lysozyme expressed in E. coli contained about 80 % of deaminated ones.2. In the case of lysozyme with the extra methionine residue at the N-terminal (M-lysozyme), the lysozyme in which more than 4 residues were deaminated (at about 50 % of total lysozyme) could not be extracted with 10 % acetic acid from the inclusion body.3. The M-lysozyme thus extracted was renaturated form the reduced form in the system of reduced an … More d oxidized glutathione, and it was found that the renaturation yield was only about 25 % and that the lysozyme in which more than 2 residues were deaminated was not renaturated but precipitated during the reaction.4. Met-lysozyme without deamidized residue was renaturated in about 50 % yield.5. New method to process the N-terminal methionine residue has been developed.6. The resulting wild type lysozyme was renaturated in 80 % yield.7. The mutant M-lysozyme in which Ala31 is replaced with Val could not be renaturated at all by the usual renaturation system was employed, but it was renaturated in about 10 % yield by the system containing 1 M urea.All of these results described above indicate that the reactions and/or mutations which reduce the solubility of denatured lysozyme, such as diaminated, addition of the hydrophobic amino acid residue at the N-terminal and the replacement of a residue with more hydrophobic one, decrease the efficiency of the renaturation of lysozyme.Thus, it is concluded that the use of E. Coli strain without deamidase, as well as the development of the method for the N-terminal methionine, is essential for the increase of the utility of the foreign protein expression system in E. Coli. Less

含有二硫键的外源蛋白在大肠杆菌中表达时，通常会形成不溶性的变性蛋白，称为包涵体。这大大降低了E.杆菌为了提高该表达系统在大肠杆菌中的实用性，杆菌为了提高该表达系统在大肠杆菌中的实用性，大肠杆菌中表达的突变型鸡溶菌酶的增溶、纯度、N-末端蛋氨酸残基的处理和复性。coli，获得了如下结果.溶菌酶在E.大肠杆菌中约80%为脱氨基的.对于N-末端有额外蛋氨酸残基的溶菌酶（M-溶菌酶），当溶菌酶中有超过4个氨基酸残基被脱氨（约占总溶菌酶的50%）时，用10%的乙酸不能从包涵体中提取.用此方法提取的M-溶菌酶，在还原型溶菌酶体系中复性， ...更多信息 d氧化型谷胱甘肽，复性率仅为25%左右，且2个以上残基脱氨基的溶菌酶在反应过程中不发生复性，而是发生沉淀.不带脱酰胺残基的Met-溶菌酶复性率约为50%.开发了一种新的处理N-末端蛋氨酸残基的方法.得到的野生型溶菌酶复性率为80%.用瓦尔取代Ala 31的突变体M-溶菌酶用通常的复性系统根本不能复性，但用含1 M尿素的系统复性，收率约为10%。在N端增加疏水氨基酸残基和用更疏水的残基取代一个残基，降低了溶菌酶的复性效率。不含脱酰胺酶的大肠杆菌菌株以及N末端甲硫氨酸方法的开发对于提高外源蛋白表达系统在大肠杆菌中的应用至关重要。杆菌少