Heterologous overexpression of a novel delta-4 desaturase gene from the marine microalga Pavlova viridis in Escherichia coli as a Mistic fusion
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Pavlova viridis, a marine alga, is rich in long chain polyunsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid (DHA). Delta-4 desaturase is the final enzyme involved in the biosynthesis of DHA in vivo. Here, a novel fatty acid desaturase gene (pkjDes4), with high similarity to functionally characterized delta-4 desaturase of Pavlova lutheri, was isolated from Pavlova viridis. During the process, a new PCR approach, designated as SEFA (self-formed adaptor PCR), combined with consecutive exploiting of splicing by overlap extension (SOE), was adopted to overcome the limitations of chromosome walking for high GC content at the 5′ terminus of the gene. The heterologous expression of the pkjDes4 gene in E. coli was carried out using Mistic, a short peptide from Bacillus subtilis, as a fusion partner. The electrophoretic band corresponding to the fusion protein was clearly visible in the whole-cell extract and membrane fractions, confirming that the expression of pkjDes4 was achieved. These results contributed to a method for isolation and overexpression of the eukaryotic membrane proteins in E. coli.
KeywordsPavlova viridis Fatty acid desaturase Mistic Membrane protein SEFA
- Neuringer M (2000) Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications. Am J Clin Nutr 71:256S–267SGoogle Scholar
- Zhou XR, Robert SS, Petrie JR, Frampton DMF, Mansour MP, Blackburn SI, Nichols PD, Green AG, Singh SP (2007) Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis. Phytochemistry 68:785–796CrossRefGoogle Scholar