一个新的蛋白质剪切系统及其剪切条件的研究

A New Protein Splicing System and its Splicing Conditions

将含麦芽糖结合蛋白－内蛋白子－几丁质结合区（简称ＭＹＢ）基因的重组质粒ｐＭＹＢ１２９转入Ｅ．ｃｏｌｉ２４２６，在ＬＢ培养基中发酵，ＩＰＴＧ低温诱导表达，菌体超声破碎，离心，上清液经直链淀粉糖亲和层析，获得ＳＤＳ－ＰＡＧＥ电泳纯的前体蛋白ＭＹＢ．ＭＹＢ中内蛋白子的Ｎ端可被还原剂ＣｙＳＨ、ＤＴＴ、β－巯基乙醇等诱导发生剪切反应．结果表明，三种还原剂中以ＤＴＴ为最佳．同时对剪切过程中温度。

For the mechanism of protein splicing,the splicing conditions for a precursor protein expressed by E.coli 2426/pMYB129 were studied.The expression product,maltose binding protein intein chitin binding domain(MYB)was purified by one step on amylose column.Though the precursor protein could self splice,it was quite stable at 4℃.However,the rate of cleavage for the precursor could be swiftly increased in the presence of reducing reagents such as DTT、CySH、β ME etc.and the velocity of cleavage was DTT>CySH>β ME in the same conditions.According to the quantity of maltose binding protein spliced at the different time on SDS PAGE,the rate constant of cleavage for DTT: k =6×10 -3 min -1 (assuming first order kinetics).Meanwhile,the rate of protein splicing was obviously affected by temperature and pH.If using the principle of protein splicing to purify recombinant proteins,it would be of some advantages as follows:(a)chemical cleavage could replace enzymatic digestion due to having a splicing site at the N terminal of Intein.(b)it could make N terminal of the target protein free,because the cleavage site of precursor was designed at C terminal of target protein.(c)cleavage could be performed on an affinity chitin column by one step.In conclusion,the system including intein CBD could become a new method in the purification of recombinant proteins.