Effects of purification tag positions on the heterologous expression of Δ6 fatty acid desaturase

Abstract:

[Background] At present, the characteristics of Δ6 fatty acid desaturase (FADS6) from various species have been identified through the yeast expression system. Since FADS6 is a multiple transmembrane protein, it is challenging to achieve large-scale expression and purification. [Objective] To construct a high-efficiency expression strategy of FADS6, the present study will analyze the influence of the location of the purification tag on heterologous expression of Mortierella alpina FADS6I (MaFADS6I). [Methods] Tandem affinity tag HRV 3C-Protein A-His was added into the Pichia pastoris vector, followed by the insertion of MaFADS6I sequence to construct recombinant vectors with the N-terminal or C-terminal tag, respectively. Recombinants were obtained through electro-transformation. The protein expression level of MaFADS6I in recombinant strains was analyzed by dot blot hybridization (dot blot), polyacrylamide gel electrophoresis (SDS-PAGE) and western blot, and the fatty acids catalyzed by MaFADS6I was detected by gas chromatography-mass spectrometry (GC-MS). [Results] Transformants with different MaFADS6I expression levels and catalytic activities were obtained. compared with the N-terminal tag, the C-terminal tag was more conducive for the expression and catalytic activity of MaFADS6I. [Conclusion] MaFADS6I with C-terminal purification tag is more conducive to the expression of the protein in the yeast system and the conversion of substrates than with N-terminal tag, providing a foundation for the high-efficiency expression and structural and functional studies of FADS6.