High level expression of bikunin in Pichia pastoris by fusion of human serum albumin

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 bikunin have been successfully cloned and expressed in E. coli and Pichia pastoris ([Fritz 1995]; [Brinkmann, Weilke et al. 1997]; [Jian-qiu, Feng-qin et al. 2008]). However, the yield of recombinant human UTI (rh-UTI) in E. coli or P. pastoris is too low and the uniform of protein doesn't to be ensured. There hasn't been report about large scale production and animal model examination so far. Therefore, the clinic value of rh-UTI is difficult to be determined all the same.Previous study showed that the use of h