PURIFICATION AND CHARACTERIZATION OF EPIA, THE PEPTIDE SUBSTRATE FOR POSTTRANSLATIONAL MODIFICATIONS INVOLVED IN EPIDERMIN BIOSYNTHESIS
PURIFICATION AND CHARACTERIZATION OF EPIA, THE PEPTIDE SUBSTRATE FOR POSTTRANSLATIONAL MODIFICATIONS INVOLVED IN EPIDERMIN BIOSYNTHESIS
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DOI：
10.1016/0378-1097(93)90535-a
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发表时间：
1993-08-15
影响因子：
2.1
通讯作者：
GOTZ, F
中科院分区：
文献类型：
--
作者：
KUPKE, T;STEVANOVIC, S;GOTZ, F
为了研究参与表皮蛋白生物合成的酶，有必要生产足够数量的前表皮蛋白(EPIA)作为底物，并设计EPIA检测系统。因此，通过雄性-EPIA融合，在大肠杆菌中表达了EPIA。纯化后的EPIA经离子喷雾质谱仪和氨基酸序列测定得到确证。对于EPIA的检测，提出了抗EPIA的抗血清。经过长时间的孵育，Xa因子不仅将EPIA从融合蛋白中切割出来，而且在R-1和I+1之间的内部切割效率也较低。通过定点突变将EPIA的内部Xa切割位点改变为-A-4-E-P-R-1-to-A-4-E-P-Q-1-来掩盖EPIA的内部切割位点。
For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A-4-E-P-R-1- to -A-4-E-P-Q-1- by Site-directed mutagenesis.