Cloning And Expression Of Pig Liver Esterase In Pichia. Pastoris

Pig liver esterase (PLE) is one of the best bio-catalysts used in chiral industry, which is extracted from pig liver previously, and full of strong capability of hydrolyzing esterases. PLE belongs to the group of enzyme of carboxylesterase (EC3. 1.1.1 ).For the pure PLE has the better activity , a procedure is designed in this research , that is to construct the engineering microorganism to express the PLE efficiently. Methylotrophic yeast expression system has been developed quickly recently. Many proteins are expressed by it in foreign countries. The protein expressed by this system can be modified correctly after the translation.Firstly, RNA is extracted from fresh tissue of pig liver , which is amplified as the templet by two groups of primers, so the aim gene is obtained successfully. Secondly, the gene and vector pPIC9K are double-enzyme cutted , which are linked by ligase in following step, and transformed into E.coli JM109. Thirdly, This new plasmid is identified by double-enzyme cutting. NcoI-cutting. PCR by different groups of primers and DNA sequencing, The results show the new plasmid pPIC9K-PLE, that is expression plasmid, is constructed successfully.After the linerization of pPIC9K-PLE, it is electrotransporated into Phichia pastoris GS115. The transformants are selected by MD plate and PCR amplification. The results show the pPIC9K-PLE is transformed successfully. The transformants are fermented and induced by methnol, and finally the expression amount of PLE is about 450mg/ml, and the MW is 60kDa or so, the activity of supernatant of fermented broth is 2.21U/ml.This research sets ground for the further study of PLE obtained in vitro.