Heterologous expression and purification of the activated siderophore synthetase PchE in Saccharomyces cerevisiae

Abstract:

[Objective] Expressing the bacterial siderophore synthetase PchE under the ADH2 promoter in Saccharomyces cerevisiae system with the PPTase Sfp from Bacillus subtilis, to explore the heterologous expression of activated bacterial protein in eukaryotic system. [Methods] The sfp gene was amplified from Escherichia coli BAP 1. Both pchE gene and pchE tandem with sfp were cloned to the yeast-E. coli shuttle vector pXW55, and transformed into the Saccharomyces cerevisiae BJ5464-npgA strain. After the purification steps including affinity chromatography and ion-exchange chromatography, HPLC was used to detect whether the PchE from E. coli and Saccharomyces cerevisiae maintain catalytic activity in the biochemical reaction in vitro.[Results] In the Saccharomyces cerevisiae expression system, prokaryotic protein PchE was obtained with high purity. And no matter assisted by the bacterial or fungal PPTase, PchE can be modified and synthesize intermediate product HPT-Cys. [Conclusion] It was firstly demonstrated that both fungal gene npgA and bacterial gene sfp can modify bacterial nonribosomal peptide synthase. With the comparison of protein expression between yeast and bacterial host, the giant protein PchE expressed by yeast has higher purity and fewer nonspecific bands. This suggested that yeast host might be more suitable for expressing and purifying functional giant protein.