Faculty of Bioindustry, Chengdu University, Waidong Shilingzhen, Chengdu, 610106, China.
AMB Express. 2012 Feb 29;2(1):14. doi: 10.1186/2191-0855-2-14.
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 Pichia pastoris and also established the purification procedure. Different fusion genes of h-UTI and domain I, domain I and domain II, domain I, domain II and domain III of human serum albumin (HSA) were inserted into expression vector pPICZαA. After expressed in shake flask, rh-bikunin was produced in an 30-L fermenter and purified by affinity chromatography and cation exchange chromatography. The final expression levels were 200 mg/L and we got totally 1.08 g (3650 IU/mg) of active purified rh-bikunin (purity is 98%) from 20 L of fermentation broth. The rh-bikunin consists of unique form with molecular masses of 25 kDa, and has the same N-terminals sequence as human native bikunin. This study provided a new method for high level expression of active rh-bikunin by using HSA as fusion parter.
纤溶酶原激活物抑制剂（Bikunin）是一种具有广谱抑制丝氨酸蛋白酶活性的蛋白聚糖，有望抑制肿瘤细胞的侵袭和转移。本研究成功地在巴斯德毕赤酵母中表达了重组人 Bikunin（rh-bikunin），并建立了纯化方法。将人血清白蛋白（HSA）的 h-UTI 与结构域 I、结构域 I 与结构域 II、结构域 I、结构域 II 与结构域 III 的不同融合基因插入表达载体 pPICZαA。经摇瓶表达后，rh-bikunin 在 30L 发酵罐中进行生产，并通过亲和层析和阳离子交换层析进行纯化。最终表达水平为 200mg/L，从 20L 发酵液中获得了 1.08g（3650IU/mg）具有活性的纯化 rh-bikunin（纯度为 98%）。rh-bikunin 具有独特的形式，分子量为 25kDa，与天然人 Bikunin 的 N 末端序列相同。本研究为利用 HSA 作为融合伴侣进行 rh-bikunin 的高效表达提供了一种新方法。
