人基质金属蛋白酶-2的表达、纯化和性质鉴定

PCR方法扩增人基质金属蛋白酶 2 (MMP 2 )不含信号肽的表达序列 ,酶切和测序鉴定正确后 ,构建酵母重组表达质粒pPIC9 MMP 2 ,电击法转化毕赤酵母 (Pichiapastoris)细胞得到阳性克隆 ,甲醇诱导获得含大量基质金属蛋白酶 2的培养上清 ,经SephacrylS 2 0 0纯化后 ,纯度达到电泳纯。明胶酶谱和SDS PAGE分析说明重组MMP 2能够降解明胶和IV型胶原 ,表明重组蛋白具有与天然MMP 2相似的底物特异性。糖基化分析和SDS PAGE表明 ,表达产物的分子量约为 5 0kD ,重组MMP 2的C 末段可能发生了降解。

Expression, Purification and Identification of Human Matrix Metalloproteinase-2

The expression sequence of matrix metalloproteinase-2 (MMP-2) has been obtained by PCR amplifying,restriction enzyme cut and sequencing analysis demonstrate that the sequence is correct.The recombinant expression plasmid pPIC9/MMP-2 containing MMP-2 is constructed and transformed the yeast %Pichia pastoris.%Recombinant matrix metalloproteinase-2 protein was expressed in %Pichia pastoris% in great deal after induction by methanol.The purity of the recombinant MMP-2 filtrated through Sephacryl S-200 reached to electrophoresis purity.With the ability to degrade gelatin and IV type collagen,recombinant MMP-2 has the similar substrate specificity with natural MMP-2.The recombinant MMP-2 with 50kD molecular weight is smaller than natural MMP-2,which suggested degradation occurred to it.