Date Published: February 29, 2012
Author(s): Xing-Hua Gou, Yu-Ying Liu, Qi-Lei Chen, Jian-Jun Tang, Da-Yu Liu, Liang Zou, Xiao-Yong Wu, Wei Wang.
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.
Bikunin, also being called urinary trypsin inhibitor (UTI), contains two antiproteolytic Kunitz domains. The protein is a proteoglycan ([Xu, Carr et al. 1998]), which has a molecular mass of about 25 kDa including a 6-7 kDa chondroitin sulfate chain ([Pugia, Valdes Jr et al. 2007]; [Chi, Wolff et al. 2008]). Bikunin is synthesized in the liver together with another plasma protein, α1-microglobulin (α1-m), forming a precursor (α1-m/bikunin precursor, AMBP). As a kind of serine proteinase inhibitor, bikunin exhibits broad inhibitory activity against many proteases, such as trypsinase, chymotrypsin, leukocyte elastase, and fibrinolytic enzyme. Moreover, human bikunin hasn’t antigenicity to human and has the characteristic of use safety, so it has been widely used as a drug for patients with acute pancreatitis, acute attack of chronic pancreatitis, acute circulation exhaustion, tumor and shock ([H Inaba 1986]; [Okuhama, Shiraishi et al. 1999]; [Kobayashi, Suzuki et al. 2003]; [Yano, Anraku et al. 2003]; [Molor-Erdene, Okajima et al. 2005]; [Qing xia 2005]; [Zhang, Liu et al. 2011]). The bikunin has many advantages such as evident effect in clinic, low side effect and low production cost. However, due to the low content in urinary, difficult collection of human urinary and high cost of purification, the bikunin is limited to apply widely. To overcome these problems, a promising alternative technique is to obtain recombinant human bikunin by gene recombination.
The expression system of yeast P. pastoris has several advantages, including the use of the alcohol oxidase I (AOX1) gene promoter, the ability of the cells to be cultivated at high density, a simplified purification procedure for secreted heterologous proteins, and modifications of foreign proteins ([Wang, Yan et al. 2008]). So, the expression system of yeast P. pastoris was used for bikunin expression. On the other hand, several parameters such as clone selection, codon optimization, fusion partners and culture conditions play a important role in obtaining high yields of protein. So, in order to optimize the level of protein production experiment, we evaluated the role of codon optimization, fusion partners and culture conditions in obtaining high yields of bikunin.
The authors declare that they have no competing interests.