乳糖诱导重组别藻蓝蛋白(rAPC)在大肠杆菌(Escherichia coli)中的表达  

EXPRESSION OF rAPC (RECOMBINANT ALLOPHYCOCYANIN) IN ESCHERICHIA COLI USING LACTOSE AS INDUCER

作　　者：唐志红[1,2] 葛保胜[1] 林凡[1] 任育红[1,3] 秦松[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学开放研究实验室 [2]烟台大学海洋学院烟台264005 [3]烟台大学生物化学系烟台264005

出　　处：《海洋与湖沼》2006年第6期524-528,共5页Oceanologia Et Limnologia Sinica

基　　金：国家863计划资助项目;2001AA6204130号;中国科学院知识创新项目;KZCX3-SW-215号。

摘　　要：采用廉价的乳糖替代IPTG诱导重组别藻蓝蛋白(rAPC)在大肠杆菌(Escherichiacoli)JM109中表达,对诱导产物表达的培养基、培养条件、诱导剂的浓度和诱导时机进行了优化,将优化条件用于5L自控发酵罐进行高密度培养。结果表明,对rAPC表达的条件进行优化后,乳糖诱导rAPC的表达率可达26.8%,与IPTG的诱导结果接近;在高密度培养中,OD600最大达21.8。研究结果可为乳糖替代IPTG大规模生产rAPC奠定基础。Phycobiliprotein is a type of photosynthetic antenna pigment found in cyanobacteria, rhodophytes, cryptophytes and certain dinoflagellates. According to their visible absorption properties, the phycobiliprotein family can be classified into four sub-families： phycoerythrocyanin, phycoerythrin, phycocyanin, and allophycocyanin. Phycobiliproteins have also been found to have anti-oxidative, anti-inflammatory, anti-tumor and cell-growth promoting activities. An APC （allophycocyanin） gene from cyanobacterium Anacystis nidulans UTEX625 was cloned, sequenced and expressed in Escherichia coli. Mouse efficacy study showed that rAPC could inhibit tumor remarkably. Isopropyl-β-D-thiogalactopyranoside （IPTG） is an effective inducer for lac promoter derived expression systems. However, IPTG is toxic and comparably expensive, which is impracticable in large-scale production of genetically engineered drugs. To discover a low-cost technique for efficient production of rAPC, in this paper, the possibility of using lactose as an inducer to substitute IPTG in the expression of rAPC in E. coli JR1 was investigated. At first, the recombinant pMAL-p2-AP （7.76kb） was transformed into E. coli JM109 （ DE3 ）. Western blot analysis revealed that the rAPC could be expressed successfully in the recombinant E. coli JR1. The growth of the recombinant E. coli and the expression of rAPC were significantly affected by cultivation media. Shake-flask cultivations in different media （ LB, 2-YT, M9 ） showed that LB medium is the most appropriate for the production of rAPC with lactose as an inducer. The final cell density OD600 reached 4.08 and the expression of rAPC amount to 21.8% of the total protein in LB medium, which was much higher than those in 2-YT and M9. Mineral salt media is often used to improve growth and the stability of plasmid, therefore, shake-flask optimization experiments were performed with different combinations of mineral salt media. Relatively good growth of the recombinant E. coli was achieved by using

关 键 词：重组别藻蓝蛋白 重组大肠杆菌 乳糖诱导 

分 类 号：Q789[生物学—分子生物学]