用定位突变探讨胰蛋白酶的稳定性

应用定位突变的方法,对鼠胰蛋白酶分子中易自溶位点的氨基酸残基进行改造,以探索提高胰蛋白酶稳定性的可能。将鼠胰蛋白酶原cDNA从质粒pTRAP上切下,插入载体M13mp8,在E.coli JM 101菌株中转化、复制后用以转染RZ1032缺陷型菌株,利用含U模板,进行按实验目的设计寡聚核苷酸引物引导的定位突变,将易自溶位点Ary105改造为Leu或Gly。经酶切和序列测定,证明在设计位点发生了预期的碱基突变。为了检查活性方便,又将含突变型胰蛋白酶cDNA从pTRAP上切下,插入另一个表达载体pTN中,转化入E.coli SM 138宿主中进行表达,表达产物分泌到围膜间隙,作专一性底物TAME活性胶方法检测,证明改造后的表达产物具有胰蛋白酶活性。对突变与野生型胰蛋白酶进行了初步比较。

Probing the Stability of Trypsin by Site-directed Mutagenesis

Attempts were made to enhance the stability of rat trypsin by replacing Arg 105, a labile site on autolysis, with Leu or Gly by site-directed mutagenesis.A BamHI-SalI fragment from the plasmid pTRAP containing the rat trypsinogen cDNA sequences was isolated and ligated into BamHI-SalI cleaved M13mp8 vector.The single-stranded uracil-containing DNA template was prepared according to the method of Kunkel(1985).Two oligodeoxynucleotides were synthesized and used as primers for site-directed mutagenesis to change Arg 105 into Leu 105 or Gly 105 Colonies from transformed E.coli JM101 were selected by digestion with restriction enzyme XhoI.The wild-type DNA contained a XhoI restriction site while the mutant did not contain it.Both the wild-type DNA and the mutant DNA were sequenced to confirm mutagenesis which had taken place at the specific site.Two mutants pTRAP(L) and pTRAP(G) were obtained.To facilitate the detection of trypsin activity, trypsin cDNAs from pTRAP(L) and pTRAP(G) were introduced into the expression vector pTN to form pTN(L) and pTN (G) which were heterologously expressed in E.coli SM138 as host.Periplasmic proteins containing trypsin secreted were fractionated by SDS-PAGE and the activity of trypsin was detected by an active polyacrylamide gel impregnated with TAME and phenol red.The appearance of yellow spots on a purple background indicates trypsin activity.Every time the yellow spot of mutant trypsin R105L appeared first and the yellow spot was larger and brighter than that of wild-type trypsin.In the case of mutant trypsin R105G, no significant difference could be detected.It appears that either the activity of the mutant trypsin R105L was increased or its stability was enhanced via protein engineering.These observations have to be confirmed by quantitative experiments on kinetics and stability properties of wild-type and mutant trypsins.