Brazzein Expression In Methylotrophic Yeast Pichia Pastoris

"Brazzein", a 54-amino-acid sweet-tasting protein, is thermostable and relatively persistent taste. Brazzein is firstly isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. The methylotrophic yeast Pichia pastoris is a developing system for high level expressing heterologous proteins in recent years,taking methanol as the unique carbon source.The plasmid pPIC9K was chosen as vector to construct expressing plasmid pPIC9K/Bi, which was linearized by restriction endonuclease BglII and transformed into the yeast Pichia pastoris GS115 by electroporation. 22 positive colonies (his+Muts ) grown on MD and MM plates as well as two colonies with high gene copies based on G418 (geneticin) resistance were screened,respectly. After cultivation and inducement of the chosen strain, the fermentation supernatant was run on Tricine-SDS-PAGE. The electrophoresis results indicated the expressed patterm and product molecular weight could be identifical to the native Brazzein. To exploite feasibility of producing Brazzein in large scale, moreover, we optimized the base salt medium that included YPG as breeding medium and fermentation medium containing 4ml/L PTM1, 6%(w/v) glycerol, 10g/L (NH4)2SO4. After optimized the culture condition was 28℃, pH 5.0 and 0.5%(V/V) methanol per 24 hours. Taken together, the secreted protein quantity under the conditions in the flask fermentation had been obviously improved. The expressed protein yield reached 385mg/L in the 10L fermentor assayed by Coomassie brilliant blue stain G-250 method. For purifing brazzein from the medium, a series of separation and purification of methods such as pI precipitation, macroporus adsorption resin, gel filtration, and ion-exchanger chromatography were performenced according to the standart protocols. It was found that the adsorption-elution effect of the strong cation exchange chromatography SP Sepharose FF was better. The elution condition was 20mmol/L,pH4.1 NaAC-HAC buffer with 0.4M NaCl for the last one. Finaly, the 600-MHZ 1H NMR spectrogram of the targe protein was drow, which in some what identifical to the diagram reported by literature. Our conclusion is the purpose protein could be expressed in Pichia pastoris, which has a good potential for application in large scale.