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...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。展开更多
We have reported that the neurotoxin released by DDT-poisoned cockroaches was identified to be tyramine, the decarboxylated product of tyrosine. The excessive production of this neurotoxin is apparently the result of ...We have reported that the neurotoxin released by DDT-poisoned cockroaches was identified to be tyramine, the decarboxylated product of tyrosine. The excessive production of this neurotoxin is apparently the result of DDT induction. Tyramine is produced by tyrosine de-展开更多
Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin...Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin. If the enzyme could react effectively at a pH higher than lysine’s pKa but lower than that of arginine, it would react more selectively with arginine-type substrates, since in that pH range, the side chain of arginie remain protonated, while that of lysine is deprotonated. For trypsin. the change of histidine (57)’s pKa reflects the shift in reaction optimum pH. Electrostatic calculations showed that when surface positive residues were mutated into neutral or negative ones, the pKa of histidine(57) would be raised and those surface charges within a cone of 70 degree around histidine(57) have strong influence on its pKa. Several sites were suggested in rat trypsin which might serve as potential mutation locations to make trypsin active at a higher pH, thus more selective towards arginine展开更多
文摘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。
文摘We have reported that the neurotoxin released by DDT-poisoned cockroaches was identified to be tyramine, the decarboxylated product of tyrosine. The excessive production of this neurotoxin is apparently the result of DDT induction. Tyramine is produced by tyrosine de-
基金Project supported by the Chinese National High Technology Development Program.
文摘Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin. If the enzyme could react effectively at a pH higher than lysine’s pKa but lower than that of arginine, it would react more selectively with arginine-type substrates, since in that pH range, the side chain of arginie remain protonated, while that of lysine is deprotonated. For trypsin. the change of histidine (57)’s pKa reflects the shift in reaction optimum pH. Electrostatic calculations showed that when surface positive residues were mutated into neutral or negative ones, the pKa of histidine(57) would be raised and those surface charges within a cone of 70 degree around histidine(57) have strong influence on its pKa. Several sites were suggested in rat trypsin which might serve as potential mutation locations to make trypsin active at a higher pH, thus more selective towards arginine
