摘要
从层理鞭枝藻藻红蓝蛋白α亚基基因表达质粒pGEMD pecA出发 ,通过酶切、削平或补齐粘末端及重新环合等技术 ,使其后的阅读框发生移码突变 ,从而得到C 端缺失 2 4个和 36个氨基酸的两个缺失突变体pGEMD pecA C2 4和 pGEMD pecA C36 .用PCR技术将已突变的基因片段转入表达载体 pET30中 ,得到pET30 pecA C2 4和 pET30 pecA C36 ,获得高效表达 .利用PCR技术从 pGEMD pecA中扩增出N 端缺失 2 0个和 32个氨基酸 pecA的突变基因片段 ,并克隆于表达载体 pET30中 ,得到pET30 pecA N2 0和 pET30 pecA N32 ,获得高效表达 .两个C 端缺失突变的脱辅基蛋白无论有或无藻红蓝蛋白连接 /异构酶催化 ,均不能与藻蓝胆素共价偶联 .两个N 端缺失突变脱辅基蛋白均能在藻红蓝蛋白连接 /异构酶催化下与藻蓝胆素共价偶联 。
With the restriction of endonuclease sites in the expression plasmid for phycoerythrocyanin α-subunit (pGEMD-pecA) ,the modified plasmids with the frameshift muations were obtained after cutting, blunt, and ligation of pGEMD-pecA. On expression of the modified plasmids gave C-terminus-deleted by 24 amino-acids and 36 amino-acids apo-proteins of phycoerythrocyanin α-subunit (PECA-C24, and PECA-C36). After amplification via PCR with certain primers, cloning, the DNA fragments were inserted into expression vector pET30, which code N-terminus-deleted by 20 amino-acids and 32 amino-acids apo-proteins of phycoery thro cyanin α-subunit (PECA-N20, and PECA-N32). Both of the C-terminus-deleted apoproteins can not be bound with phycocyanobilin in spite of the presence or absence of phycoerythrocyanin lyase/isomerase. Both of the N-terminus deleted apoproteins can be bound with phycoviolobilin converted in situ from phycocyanobilin in the presence of phycoerythrocyanin lyase/isomerase.
出处
《华中科技大学学报（自然科学版）》
EI
CAS
CSCD
北大核心
2002年第8期110-113,共4页
Journal of Huazhong University of Science and Technology(Natural Science Edition)
基金
国家自然科学基金资助项目 ( 39770 1 75 )
湖北省科技攻关课题资助项目 ( 2 0 70 0 0 331 )
关键词
藻红蓝蛋白α亚基
分子设计
重组
层理鞭枝藻
连接/异构酶
phycoerythrocyanin α-subunit
molecular design
reconstitution
Mastigocladus laminosus
lyase/isomerase
