Expression and purification of active recombinant human bikunin in Pichia pastoris.
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.
Jian-qiu
Wang
W
Department of Biological Engineering, College of Pharmacy, Jilin University, 1163 Xin Min Street, Changchun 130021, PR China.
Feng-qin
Yan
Y
Dou-dou
Wang
W
Lei
Ban
B
Nan
Sun
S
Cong-yan
Li
L
Ting
Zhang
Z
Wei-qun
Yan
Y
D000510:Alpha-Globulins / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D014361:Trypsin Inhibitors
D000510:Alpha-Globulins; D017931:DNA Primers; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C016001:alpha-1-microglobulin