High
Expression of LIP1 in Pichia pastoris
BEI Jin-Long, WANG Jin-Wen,
WANG Xun-Zhang*, LONG Qin-Xing, YANG Lin, DENG Ying-Yu
( The State Key Laboratory for Biological Control,
Biopharmaceutical Center, Zhongshan University, Guangzhou 510275, China )
Abstract The gene of LIP1, the most important
isoenzyme of Candida rugosa lipase (CRL), was artificially synthesized
according to its mature peptide sequence. It consisted of 20 codons of
preference in Pichia pastoris. The artificial gene was cloned into
methanol-inducible expression vector pPICZaA, and constitutive expression
vector pGAPZaA, respectively. The linearized recombinant plasmids were
transformed into chromosome of Pichia pastoris SMD1168H strain by
electroporation. The abilities of expressing LIP1 in both transformed yeasts
had been compared, and the yeast transformed with pGAPZaA was more efficient than
pPICZaA. A recombinant yeast strain named CHT-II expressed LIP1
constitutively, and was the most efficient one. Some enzymetic properties of
the recombinant LIP1 were also determined. CHT-II secreted LIP1 into supernate
at a level of 2.00´105 u/L after 72 h (the cells had been
transferred to fresh culture medium at the 24th h). After optimizing the
conditions for high cell-density fermentation, the selected yeast strain could
secrete LIP1 into supernate at a level of 1.395´106 u/L after 72 h. The
results indicated that this modification of lip1 gene was successful.
Key
words
artificial synthesis; CRL; LIP1; Pichia pastoris; constitutive
expression
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