Expression, purification and application discussion of red fluorescent protein variant mCherry

Abstract

Objective To express red fluorescent protein variant mCherry with pET plasmid in E. coli, and analyze the relationship of mCherry expression with its fluorescent characteristics, providing basis for mCherry purification and its application as prokaryotic report system. Methods The coding sequence was chemically synthesized according to the amino acid sequence of mCherry from GenBank. The constructed expression vector pET-mCherry was transformed into E. coli BL21(DE3). Fluorescent intensity, expression amount and leakage into medium were analyzed at different inducing time points. Bacteria were collected and resuspended in PBS after 10 h induction. Recombinant mCherry was extracted by continuous stirring process. The recovery rate of mCherry was estimated by fluorescence assay, and the purity was analyzed in gel electrophoresis. Results The expression vector pET-mCherry was successfully constructed and recombinant mCherry was expressed efficiently upon induction. There was a time lag between fluorescence detection and mCherry expression. Recombinant mCherry continuously leaked into medium. After one-step extraction, the product was obtained with purity above 95%. Conclusions mCherry can be efficiently expressed in pET system, and overexpressed mCherry can leak into medium following induction, favoring purification. Culture time would be the key factor when mCherry is used as prokaryotic report gene. Key words: Escherichia coli; Luminescent proteins; Cloning, molecular; Gene expression; Preparation; Red fluorescent protein mCherry