Jian-qiu Wang, Feng-qin Yan, Dou-dou Wang, Lei Ban, Nan Sun, Cong-yan Li, Ting Zhang, Wei-qun Yan
Department of Biological Engineering, College of Pharmacy, Jilin University, 1163 Xin Min Street, Changchun 130021, PR China.
Protein Expr Purif. 2008 Aug;60(2):127-31. doi: 10.1016/j.pep.2008.03.025. Epub 2008 Apr 4.
Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification procedure. The cDNA encoding human bikunin was cloned by PCR and inserted into the expression vector pPICZalphaC. After expressed in shake flask, rh-bikunin was produced in an 80-L fermenter and purified by cation exchange chromatography and reverse phase chromatography. The rh-bikunin was active by trypsin inhibition test. The final expression levels were 55 mg/L and we got totally 1.44 g (5600 inhibitor units/mg) of purified rh-bikunin (purity is 95%) from 40 L of fermentation broth. The rh-bikunin consists of two forms with molecular masses of 24 and 21 kDa, respectively. Both forms were immunoreactive by Western blotting and N-terminals were correctly processed by amino-terminal sequencing. This study provided a new method for expression and purification of active rh-bikunin.
比库宁是一种蛋白聚糖，对丝氨酸蛋白酶具有广谱抑制活性，可能抑制肿瘤细胞的侵袭和转移。在此，我们已在甲基营养型酵母毕赤酵母中成功表达重组人比库宁（rh-比库宁）并建立了纯化程序。通过PCR克隆编码人比库宁的cDNA，并将其插入表达载体pPICZalphaC中。在摇瓶中表达后，rh-比库宁在80-L发酵罐中产生，并通过阳离子交换色谱和反相色谱进行纯化。通过胰蛋白酶抑制试验检测，rh-比库宁具有活性。最终表达水平为55 mg/L，从40 L发酵液中我们共获得1.44 g（5600抑制剂单位/mg）纯化的rh-比库宁（纯度为95%）。rh-比库宁由两种形式组成，分子量分别为24 kDa和21 kDa。两种形式通过蛋白质免疫印迹均具有免疫反应性，并且通过氨基末端测序正确处理了N末端。本研究为活性rh-比库宁的表达和纯化提供了一种新方法。
