World Journal of Microbiology and Biotechnology

, Volume 27, Issue 12, pp 2931–2937 | Cite as

Heterologous overexpression of a novel delta-4 desaturase gene from the marine microalga Pavlova viridis in Escherichia coli as a Mistic fusion

Original Paper

Abstract

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.

Keywords

Pavlova viridis Fatty acid desaturase Mistic Membrane protein SEFA 

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.State Key Lab of Microbial TechnologyShandong UniversityJi NanPeople’s Republic of China
  2. 2.Molecular Genetics Lab, School of Life ScienceTsinghua UniversityBeijingPeople’s Republic of China

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