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�й����ﻯѧ���������ѧ�� 2004, Vol. 20 Issue (01 ):73-73     DOI:
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Site-directed Mutation of Violaxanthin De-epoxidase from Tea Plant (Camellia sinensis) in vitro and Expression, Bio-activity Assay of the Mutants
WEI Chao-ling 1),2), JIANG Chang-jun 1)*, TAO Han-zhi 1), WAN Xiao-chun 1) ( 1)Key Laboratory of Tea Biotechnology, Ministry of Agriculture, Anhui Agricultural University, Hefei 230036,China; 2)Department of Agronomy, Anhui Agricultural University, Hefei 230036, China)

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ժҪ ��Swiss Prot��������ݿ��в�ѯ���ֲ����ϻ����ѻ�����ø (VDE)���׽ṹ�к���lipocalin������ ,�����ص����취�Ѹ�����������ص�Gly(GGG)��Trp(TGG)�ֱ𶨵�ͻ��ΪLeu(TTG)��Tyr(TAG) .�����˱������pET 32a G��L��pET 32a W��Y ,��E .coliBL2 1trxB(DE3)�������յ���� ,���Ա�ﵰ�׽���His Tag�׺ʹ��� .������ ,��ﵰ�׵ķ�������Ԥ�Ƶ���� ,Ϊ 5 8 9kD ,�����ռ�����ܵ��׵� 4 5 % .����ø�ٷ�Ӧ�����ó� ,G��L��W��Yͻ�䶼�ܵ��²���VDE������Դ��Ƚ��� ,֤����lipocalin��������VDE����Ҫ��������֮һ .
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Abstract�� The lipocalin signature of violaxanthin de-epoxidase(VDE) from camellia sinensis was found in Swiss-Prot protein database. Gly(GGG) and Trp(TGG), the most conserved sites, in lipocalin signature were site-mutated to Leu(TTG) and Tyr(TAG), respectively, by using over-lap extension. The expression plasmids, pET-32a/G��L and pET-32a/W��Y, were expressed in E.coli BL21trxB(DE3) by IPTG mduction, then the expressed products were purified by Talon Metal Affinity Resin and determined by bio-activity assay. The results showed that the molecular mass of expressed protein was 5.89 kD as expected, and the amount of expression accounted for 45% of the total bacterial protein. Compared with the wild type VDE of camellia sinensis, the bio-activity of G��L mutant or W��Y was very low. It was demonstrated that lipocalin signature was one of the most active domains in VDE.
Keywords�� Camellia sinensis   violaxanthin de-epoxidase   site-directed mutation   expression   activity assay      
Received 2003-02-26;
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Τ����.�����ϻ����ѻ�����ø��������ⶨ��ͻ�估��ͻ����ı��ͻ��Լ� [J]  �й����ﻯѧ���������ѧ��, 2004,V20(01 ): 73-73
.Site-directed Mutation of Violaxanthin De-epoxidase from Tea Plant (Camellia sinensis) in vitro and Expression, Bio-activity Assay of the Mutants [J]  Chinese Journal of Biochemistry and Molecular Biol, 2004,V20(01 ): 73-73
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http://cjbmb.bjmu.edu.cn/CN/     ��     http://cjbmb.bjmu.edu.cn/CN/Y2004/V20/I01 /73