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Expression and purification of active recombinant human bikunin in Pichia pastoris.

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 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Jian-qiu W,Feng-qin Y,Dou-dou W,Lei B,Nan S,Cong-yan L,Ting Z,Wei-qun Y

doi

10.1016/j.pep.2008.03.025

subject

Has Abstract

pub_date

2008-08-01 00:00:00

pages

127-31

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(08)00095-8

journal_volume

60

pub_type

杂志文章

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