Abstract
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.
Highlights
Bikunin, being called urinary trypsin inhibitor (UTI), contains two antiproteolytic Kunitz domains
Previous study showed that the use of human serum albumin (HAS) as N-terminal fusions can be an effective technique to express difficult proteins in mammalian cells ([Carter, Zhang et al 2010]; [Zhang, Carter et al 2010])
Fusion genes of h-UTI and domain I, domain I and domain II, domain I, domain II and domain III of human serum albumin were inserted into expression vector pPICZaA, respectively
Summary
Being called urinary trypsin inhibitor (UTI), contains two antiproteolytic Kunitz domains. Bikunin is synthesized in the liver together with another plasma protein, a1microglobulin (a1-m), forming a precursor (a1-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. The bikunin has many advantages such as evident effect in clinic, low side effect and low production cost. 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
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