Expression, Purification And Bioassay Of Mleptin

Leptin is an adipocyte-derived hormone coded by the obese gene(OB) that acts as amajor regulator for food intake and energy homeostasis. Escherichia coli BL21andPichia pastoris GS115expression systems for mleptin were studied in the thesis and theresults showed the amount of the recombinant mleptin reached12g/L in buffalo flaskby using the E coli. BL21system. The average recovery rate of active mleptin improvedgreatly because of using slow dilution dialysis. As to the low expression efficiency inGS115expression system, GS115proteomics differences cultured in different carbonsource mediums were studied. The findings will be helpful to develop novel strainswhich may optimize the Pichia pastoris heterologous protein expression system.Escherichia coli BL21expression system: The correct constructed recombinantplasmid PET-28a-His-mleptin was transformed to E.coli BL21induced by IPTG and theresult showed the target protein mainly exsisted in inclusion bodies. The best inductionconditions for the target protein are:0.1mmol/L IPTG、induction time6h and inductiontemperature37℃.The amount of the recombinant mleptin expressed in E. coli incommon flask was about10g/L while reached about12g/L in buffalo flask. Afterproper denaturation, renaturation and gel chromatography through a Ni2+affinitycolumn, about5.2g pure active mleptin with a purity of being above95%was isolatedper liter with an average recovery rate43.3%. The C57/BL6mice were treated with thepurified product through intraperitoneal and the body weight、food intake wereanalyzed. The result showed that the body weight and the food intake of theexperimental group significantly reduced compared to the control group following ICVinjection so the purified recombinant protein was proved to be biologically active.GS115eukaryotic expression system: The recombinant plasmid pPICZ α-His-mleptin was transformed to competent cell GS115by electroporation induced bymethanol. The result showed the expression efficiency of the target protein is so low inGS115expression system that it can’t be used for scale production. By using LC-ESI-MS/MS combined with Griffin’s Label-free normalized quantification method,intracellular and supernatant protein profiling and protein abundance of GS115in4 different carbon source mediums were identified and compared. The findings will behelpful to develop novel strains which may optimize the Pichia pastoris heterologousprotein expression system.