Production of recombinant EGFP via surface display of ice nucleation protein and self-cleavage intein

Published on May 1, 2011in Best Practice & Research Clinical Haematology1.99
· DOI :10.1016/j.bej.2011.02.009
Jiun-Yan Wu4
Estimated H-index: 4
(NCHU: National Chung Hsing University),
Tsung-Yu Tsai1
Estimated H-index: 1
(NCHU: National Chung Hsing University)
+ 5 AuthorsYung-Chuan Liu24
Estimated H-index: 24
(NCHU: National Chung Hsing University)
Abstract In this study, a novel recombinant protein production system was developed by constructing a protein chimera with a self-cleavage segment along with a cell surface display segment to finally produce a model protein entity ( i.e. , enhanced green fluorescent protein, EGFP). In the plasmid construction, the EGFP gene was fused to genes of a self-cleaving intein (INT) and an ice nucleation protein (INP) which can anchor on the cell membrane. In the cultivation of recombinant Escherichia coli , the cells expressed high green florescence after the addition of isopropyl-β- d -thiogalactopyranoside (IPTG) as the inducer. By simply holding the cell pellets in Tris–HCl (pH 10.0) buffer at room temperature, EGFP was solubilized from the INP–INT segment embedded on the cell surface via intein's self-cleavage function. The EGFP concentration of 273 mg/L and recovery of 88% were obtained at day 5, whereas the best EGFP productivity of 187 mg/L/d was obtained at day 1. The EGFP can be harvested only via centrifugation, and no cell disruption process is required. This simplified approach is expected to be applicable for obtaining recombinant functional proteins for academic and industrial use.
  • References (23)
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