Department of Bioscience & Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian, China.
Appl Microbiol Biotechnol. 2010 Jan;85(3):635-42. doi: 10.1007/s00253-009-2141-2. Epub 2009 Jul 29.
Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40 degrees C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-L: -phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.
蛇岛蝮蛇来源的类凝血酶 Gloshedobin 通常以包涵体的形式在大肠杆菌细胞中表达。在这项工作中，Gloshedobin 的 N 端分别与三个融合伴侣 NusA、GST 和 TrxA 融合，并在大肠杆菌中表达为融合蛋白。结果表明，NusA 是提高重组 Gloshedobin 可溶性的最有效融合伴侣。纯化的 NusA 融合 Gloshedobin 的总收率为 64.6%，在 SDS-PAGE 凝胶中显示为一条带，分子量约为 90 kDa。重组产物均具有纤维蛋白原凝固和纤维蛋白原水解活性。在最佳条件（pH8.0 和 40°C）下，纯化的 NusA 融合 Gloshedobin 的酰胺酶活性为 506 U/mg。NusA 融合 Gloshedobin 对各种抑制剂的抑制研究表明，丝氨酸蛋白酶抑制剂苯甲基磺酰氟和 N-甲苯磺酰-L-苯丙氨酸氯甲基酮强烈抑制其酰胺酶活性，而乙二胺四乙酸以及肝素和水蛭素则不抑制，表明 NusA 融合 Gloshedobin 表现出与天然 Gloshedobin 相同的特征。这项工作的策略可能有助于提高蛇毒中其他类凝血酶在大肠杆菌中的可溶性表达水平。
