摘要： 从经水杨酸处理的拟南芥开花期植株中获得cDNA,扩增得到Alpha-dioxygenase 1(DOX1)基因,进行原核表达、纯化和生物活性检测。利用原核表达载体pMAL-c4x在T7 Express CompetentE.coli和BL21(DE3)-RIPL codon+菌株中表达DOX1,经Amylose Resin亲和层析柱纯化。SDS-PAGE结果表明,重组融合蛋白在BL21(DE3)-RIPL codon+中的表达通过灰度值比较分析以可溶性为主,表达率约3.7%,纯化的DOX1纯度可达45%。愈创木酚法表明,可溶性重组蛋白不具有过氧化物酶活性;2,4-DNP法测试表明可溶性重组蛋白具有脂肪酸双加氧酶活性。
关键词:
Alpha-dioxygenase1,
重组表达,
2,
4-DNP,
亲和纯化,
活性
Abstract: Recombinant DOXI was cloned into pMAL-c4x expression vector and expressed in E. coli T7 ExpressCompetent E. cola and BL21( DE3) RLPL codon+.Purified using amylose resin column, a fusion protein about 114 kD wasdetected by SDS-PAGE in the IPTG induced recombinant BL21(DE3) RIPL codon+ strain，and it's yield acounts for3. 7% of the total bacterial proteins, the purity of recombinant proteinwas about 45%.The soluble fusion protein had un-detectable peroxidase activity by the guaiacol methol;The alpha dioxygenase activity of the purified protein was tested us-ing 2, 4 DNP，result showed that the soluble fusion protein had detectable alpha-dioxygenase activity.
Key words:
Alpha-dioxygenase 1,
Recombinant expression,
2,4-DNP,
Affinity purification,
Activity
中图分类号:
张小梅, 韩念法, 张美祥, 李广录, 范三红, 郭蔼光. 拟南芥Alpha-dioxygenase 1的原核表达、纯化及活性的检测[J]. 华北农学报, 2008, 23(3): 9-12. doi: 10.7668/hbnxb.2008.03.003.
ZHANG Xiao-mei, HAN Nian-fa, ZHANG Mei-xiang, LI Guang-lu, FAN San-hong, GUO Ai-guang,. Expression, Purification and Activity Analysis of Arabidopsis-dioxygenase 1[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2008, 23(3): 9-12. doi: 10.7668/hbnxb.2008.03.003.