Pálsdóttir Helga Margrét, Gudmundsdóttir Agústa
Science Institute, University of Iceland, Laeknagardi, Vatnsmýrarvegi 16, Reykjavík IS-101, Iceland.
Protein Expr Purif. 2007 Feb;51(2):243-52. doi: 10.1016/j.pep.2006.06.008. Epub 2006 Jun 23.
The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.
最近分类的III组胰蛋白酶包括大西洋鳕鱼（Gadus morhua）胰蛋白酶Y以及来自其他冷适应鱼类物种的七种类似物。这八种III组胰蛋白酶已通过其cDNA和推导的氨基酸序列进行了表征，但尚未从其天然来源中分离出任何一种酶。本研究描述了重组HP-硫氧还蛋白-胰蛋白酶Y融合蛋白在His-Patch硫氧还蛋白大肠杆菌表达系统中的成功表达和纯化，以及通过色谱方法对其进行的纯化。胰蛋白酶Y的重组形式先前已在毕赤酵母中表达，使其成为第一个经过生物化学表征的III组胰蛋白酶。它对胰蛋白酶和胰凝乳蛋白酶底物具有双重底物特异性，并在2至21摄氏度之间的温度下表现出增加的活性，在30摄氏度时完全失活。该研究的目的是通过找到一个比我们在毕赤酵母系统中能够获得的产量更高的酶表达系统，来促进对重组胰蛋白酶Y的进一步研究。此外，胰蛋白酶Y的商业生产将需要一个高效且廉价的表达系统，如本文所述的His-Patch硫氧还蛋白大肠杆菌表达系统，因为该酶在大西洋鳕鱼中的产量非常低。
