Expression of a Beauveria bassiana chitinase (Bbchit1) in Escherichia coli and Pichia pastoris.
Beauveria bassiana chitinase (Bbchit1) is an important cuticle degrading enzyme involved in pathogenesis of fungi against insect. To obtain enough active chitinase for performing in vitro functional analysis, Bbchit1 gene was expressed in Escherichia coli and Pichia pastoris, respectively. The high-level production of recombinant Bbchit1 was detected in E. coli expression system, however mainly located in inclusion bodies. Refolding of solubilized inclusion body proteins was achieved by dialysis. In P. pastoris expression system, Bbchit1 was secreted into the culture medium under the induction of methanol. Active Bbchit1 was purified to near 90% purity from culture medium by desalting chromatography and anion exchange chromatography. The yield of Bbchit1 produced by P. pastoris was estimated at 153 mg/L, significantly higher than that of the refolded Bbchit1 from E. coli inclusion bodies (50 mg/L). Additionally, the specific activity of Bbchit1 from P. pastoris was also higher than that from E. coli (3.9 U/mg versus 2.8 U/mg). These results indicated P. pastoris was a convenient expression system for the efficient production of Bbchit1.
Fan
Yanhua
Y
Biotechnology Research Center, Southwest University, Beibei, 400716 Chongqing, PR China.
Zhang
Yongjun
Y
Yang
Xingyong
X
Pei
Xiaoqiong
X
Guo
Shujuan
S
Pei
Yan
Y
D052982:Beauveria / Q000201:enzymology*; D002688:Chitinases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D010843:Pichia / Q000378:metabolism*; D017510:Protein Folding
D002688:Chitinases