| Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli | |
Cao, Yujin; Xian, Mo ; Yang, Jianming; Xu, Xin; Liu, Wei; Li, Liangzhi
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| 2010-02-01 | |
| 发表期刊 | PROTEIN EXPRESSION AND PURIFICATION
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| ISSN | ISSN:1046-5928 |
| 卷号 | 69期号:2页码:209-214 |
| 摘要 | Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (M). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS-PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1 Delta 9) and cis-vaccenate (18:1 Delta 11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1 Delta 9)was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase.; Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (M). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS-PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1 Delta 9) and cis-vaccenate (18:1 Delta 11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1 Delta 9)was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase. (C) 2009 Elsevier Inc. All rights reserved. |
| 文章类型 | Article |
| 关键词 | Gc-ms Heterologous Expression Ssi2/fab2 Stearoyl-acp Desaturase |
| 学科领域 | 生物基化学品 |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| DOI | 10.1016/j.pep.2009.08.011 |
| 关键词[WOS] | FATTY-ACID DESATURASE ; MEMBRANE-LIPIDS ; COA DESATURASE ; GENE ; IDENTIFICATION ; CLONING ; PLANTS ; BIOSYNTHESIS ; PURIFICATION ; MUTANTS |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS类目 | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS记录号 | WOS:000272653300013 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/450 |
| 专题 | 生物基材料组群 |
| 作者单位 | Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, Qingdao 266101, Peoples R China |
| 推荐引用方式 GB/T 7714 | Cao, Yujin,Xian, Mo,Yang, Jianming,et al. Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli[J]. PROTEIN EXPRESSION AND PURIFICATION,2010,69(2):209-214. |
| APA | Cao, Yujin,Xian, Mo,Yang, Jianming,Xu, Xin,Liu, Wei,&Li, Liangzhi.(2010).Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli.PROTEIN EXPRESSION AND PURIFICATION,69(2),209-214. |
| MLA | Cao, Yujin,et al."Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli".PROTEIN EXPRESSION AND PURIFICATION 69.2(2010):209-214. |
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