Yu Xueling, Li Zhaofa, Xia Xiaobing, Fang Hongqing, Zhou Changlin, Chen Huipeng
Beijing Institute of Biotechnology, 20 Dong Da Street, Fengtai District, Beijing 100071, China.
Protein Expr Purif. 2007 Oct;55(2):257-61. doi: 10.1016/j.pep.2007.07.002. Epub 2007 Jul 14.
Ancrod is known as a thrombin-like enzyme from the venom of Calloselasma rhodostoma. The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPIC9 and was subsequently expressed in the yeast Pichia pastoris. Recombinant ancrod was produced in 5-L bioreactor using a sorbitol-methanol feeding strategy and recovered from the fermentation broth by hydrophobic, affinity, and ion exchange chromatography. SDS-PAGE analysis revealed that ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43-48 kDa which decreased to about 29 kDa after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein.
安克洛酶是一种从红口蝮蛇毒液中提取的类凝血酶。编码安克洛酶的cDNA采用酵母偏好密码子合成，并插入真核表达载体pPIC9中，随后在毕赤酵母中表达。重组安克洛酶在5升生物反应器中采用山梨醇 - 甲醇补料策略生产，并通过疏水色谱、亲和色谱和离子交换色谱从发酵液中回收。SDS - PAGE分析表明，安克洛酶存在异质性糖基化，预期分子量为43 - 48 kDa，用N - 糖苷酶F去糖基化后降至约29 kDa。测定了重组安克洛酶的纤维蛋白原溶解活性和酶谱活性，发现其与天然蛋白相似。
