| Apolipoprotein C-Ⅱ(ApoC-Ⅱ) as one of the most important ApoC tribe subtype, play an important role in the Lipid metabolism in the medium-term, it is the lipoprotein lipase (LPL)auxiliary factor and can activate the multiple sources of LPL, ApoC-Ⅱalso can reduce lactate dehydrogenase (LDH) release, prevented the morphological changes of cells, protect LDH-induced damage to endothelial cells and play an important role in preventing the formation of atherosclerosis. All these demand for a substantial increase in ApoC-Ⅱ,However, it usually be extracted from human plasma separation,which source is extremely limited, obtain ApoC-Ⅱhas become research and application bottlenecks. Therefore, using genetic engineering methods to produce recombinant human ApoC-Ⅱ(rhApoC-Ⅱ) is of great significance.1 Pichia pastoris Engineering Strain ConstructionThe linearized recombinant expression plasmid pPICZαC-ApoC-Ⅱwere introduced into P. pastoris X-33 by electroporation using a Micropulser. Transformed cells were selected by growth on yeast extract peptone dextrose (YPD) agar plates containing zeocin. After the multicopy transformants appeared, 30 colonies were cultured in shake flask, genomic DNA was isolated from different clones and integration was confirmed by PCR. And the expression level of rhApoC-Ⅱwas evaluated by TRICINE-SDS-PAGE analysis. The experimental results show that rhApoC-ⅡPichia pastoris expression system can be induced by methanol to secrete rhApoC-Ⅱ. rhApoC-Ⅱexpression began on the 1st day, after 4 days, rhApoCⅡreached the top level. On the fifth day, it began to decline. And the stained bands can be seen in the corresponding molecular weight 8.9 kD.2 rhApoCⅡPichia Yeast Expression OptimizationPichia yeast can growth well under the conditions of pH3.0 ~ 6.0 which offerd facilitate to optimize the fermentation conditions. High expression of the selected strains were inoculated into pH3.2, pH3.6, pH4.0, pH4.4, Ph4.8, pH5.2, pH5.6, pH6.0 medium in the fermentation, expression products were tested by TRICINE-SDS-PAGE. The results showed that, under the condition pH5.6 selected strain expressed the highest production.3 rhApoC-ⅡPichia yeast expression under the optimized conditions in 2L BMMYAmplificate high expression rhApoC-Ⅱengineering strain under the optimized conditions in 2L BMMY, TRICINE-SDS-PAGE tested the expressed product, results show that in the methanol induction conditions, the engineered bacteria stability express rhApoC-Ⅱ. 4 rhApoC-ⅡPichia yeast large-scale fermentation and purificationA stock culture of recombinant P. pastoris X33- ApoCⅡwas grown to an OD600 of 8 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. The glycerol phase lasted 24 h and the methanol feed started when the culture reached an OD600 of 95 and wet weight of 180 g/L. Under optimal conditions (pH 5.6, 28℃, DO set at 25-30% and the supply speed of methanol is 11ml/h/L initial fermentation volume), an OD600 of 143 (corresponding to a wet weight of 274g/L) was obtained after 12h of induction. The rhApoC-Ⅱsolution was purified with cation exchange chromatography (SP Sepharose XL) and reverse phase chromatography (Source 30). Following these processes, we could get 138.6mg purified rhApoC-Ⅱfrom 30 L culture medium.
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