| In 1977, a kind of acid glycoprotein whose molecular weight is 46 kD was found, and that acid glycoprotein was named Apolipoprotein A-Ⅳ(apoA-Ⅳ). It contains 396 amino acids, with glycine as N-terminal and lysine as C-terminal. It is a part of CM, HDL and VLDL. apoA-Ⅳis synthesized by small intestina, and regulated by blood levels of triglycerides and cholesterol. After synthesized by small intestina, apoA-Ⅳis transfered into the blood circulation as chylomicrons through intestinal lymphatic system, or come into the blood circulation directly. apoA-Ⅳcan regulate lipid metabolism by redistribution between apolipoproteins under different conditions.The main physiological functions of apoA-Ⅳare as follows:①ApoA-Ⅳcan causes a reduction of feeding;②ApoA-Ⅳcan participate in blood fats transportation and metabolism;③ApoA-Ⅳcan prevent the occurrence of artery atherosclerosis.1 The establishment of recombinant apoA-ⅣPichia pastoris eukaryotic expression systemThe linearized recombinant expression vectors were introduced into Pichia pastoris X-33 by electroporation using a Micropulser. With the application of specific expression primers to identify pPICZα-apoA-Ⅳthe result showed that a product of 1155 bp was amplified in some clones. The supernatants were harvested to run on the SDS-PAGE. The results indicated that the protein, whose molecular weight was almost46 kD, was expressed which is in accordance with the anticipated size.2 The expression of recombinant apoA-Ⅳin Pichia pastoris eukaryotic expression systemCultivated the higher expression Pichia pastoris strain in BMGY (BMMY) medium, sampled 1 mL broth at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, supernatants were run on SDS-PAGE and ELISA. The results showed that The Pichia pastoris started to express apoA-Ⅳwith the vector signal peptide and inductiong of methanol at the time of 24 h. the expression level get its maximum at the time of 96 h and there is a platform phase between the time of 96 h and 120 h. Then The expression level of recombinant apoA-Ⅳbegin to decrease and the expression level of contaminate protein began to increase. So the best time to collect the fermentation broth is 96 h.Cultivated the higher expression Pichia pastoris strain in BMGY (BMMY) medium whose pH is 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 ,7.0 and 7.5. After the induction of 96 h, the supernatants were run on the SDS-PAGE and ELISA. The results showed that the secretion of recombinant protein had a highest yield at pH 6.5.Through the Mono Q HR5/5 anion column chromatography of ?KTA explorer 100, we determined that pH 8.0 is the optimum protein adsorption environment and the corresponding elution NaCl concentration is 0.32 mol·L-1. we added 0.1% TFA to the elution fractions containing the fusion protein and load the sample to the reversed-phase hydrophobic chromatography SourceTM30 RPC HR 10/30. The result showed that 75% methanol (containing 0.1% TFA) can elute the protein sufficiently; methanol and TFA can be removed by rotary evaporation.3 large-scale fermentation process of recombinant apoA-ⅣPichia pastoris has many advantages as a kind of expression host, and it's very suitable for large-scale expression of the extraneous proteins. A stock culture of recombinant P. pastoris X33-apoA-Ⅳwas grown in a 5-L shake flask containing 2 L YPD. The shake-flask culture was used to inoculate a 80 L fermenter containing 30 L of fermentation basal salts medium FM21 supplemented with PTM1 trace saltsand biotin. And the best pH is 6.5, DO between 25%～30% and the supply speed of methanol is 330 mL·h-1 initial fermentation volchaology.The recombinant apoA-Ⅳexpression supernatant was concentrated by ultrafiltration (vivaflow 200) , and purified with anion exchange chromatography (Q Sepharose XL) and reverse phase chromatography (SourceTM30). Following these processes, we could get 27.37 mg·L-1 of purified recombinant apoA-Ⅳfrom 30 L culture medium. And the degree of purity could be more than 94.6% and the yield was about 44%.
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