别藻蓝蛋白基因在毕赤酵母(Pichia pastoris)中的表达

EXPRESSION OF ALLOPHYCOCYANIN GENE IN PICHIA PASTORIS

采用亚克隆方法,将别藻蓝蛋白(allophycocyanin,APC)基因插入到毕赤酵母整合型表达载体pPIC6αA中,再用SacⅠ线性化,纯化后,用氯化锂法转化毕赤酵母菌株X-33,筛选表达APC的重组子。PCR和序列分析表明,APC基因已整合到酵母染色体中。重组子经甲醇诱导,用Western blotting在培养基上清液检测到APC,表明别藻蓝蛋白(APC)在毕赤酵母中可以表达。APC由两种亚基组成,分子量分别为19kDa和21kDa。重组子经摇瓶培养48h表达量达4.4mg/L。

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. The allophycocyanin gene was transferred into Escherichia coli in our laboratory. The recombinant APC has high expression levels and anticancer and immunity-enhancing effects. The methylotrophic yeast, Pichia pastoris has proved to be an outstanding host for high-level production of both secreted and intracellular proteins. The present study is to construct allophycocyanin expression vectors of P. pastoris and express new recombinant APC in order to screen stronger antitumor protein. A DNA fragment coding APC was obtained from plasmid pMAL-p2/APC digested by EcoR I and cloned into the EcoR I site of the vector pPIC6α A. The constructed expression plasmid pPIC6αA/APC digested by EcoR I was analyzed with agarose gel electrophoresis and PCR. The result showed that the correct Open Reading Frame of APC was obtained. The constructed expression plasmid was digested by Sac I. The linearized DNA was transformed into P. pastoris strain X-33 by Lithium Chloride. The transformants were selected on YPD plates containing the appropriate concentration of blasticidin. The genomic DNA isolated from the recombinant X-33 strain was used for PCR. The PCR product was detected on 1% agarose electrophoresis gel. Two bands at 0.5kb （using primer APP1 and APP2） and 1.3kb （using primer APP1 and primer 3＇ AOX1） in size were shown, which correspond to the size of alpha subunit of APC and APC respectively. Bigger fragments were purified and sequenced. The result showed the intact nucleotide sequence of APC had integrated into the yeast genome. The nucleotide sequence of APC gene consisted of 996bp including 510bp of the alpha subunit and 486bp of beta subunit. The content of G＋C is 56.4% for subunit and 56.1% for 13 subunit. APC consisted of 330 amino acids. The expression of rAPC in BMMY medium was determined by ELISA method. The highest expression yield of rAPC in the BMMY medium was 4.4mg/L at 48h. The Western blot analysis of the cultured supernatant of the yeast indicated that the recombinant allophycocyanin was expressed. APC consisted of two kinds of subunits with the molecular weight of 21kDa and 19kDa respectively.