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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
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)
  • Citations (5)
References23
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#1Maxim Zakhartsev (Alfred Wegener Institute for Polar and Marine Research)H-Index: 10
#2Carmen Momeu (JU: Jacobs University Bremen)H-Index: 4
#1Philip Shemella (RPI: Rensselaer Polytechnic Institute)H-Index: 7
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#1Po-Hung Wu (NTHU: National Tsing Hua University)H-Index: 1
#2R. Giridhar (NCKU: National Cheng Kung University)H-Index: 4
Last.Wen-Teng Wu (NCKU: National Cheng Kung University)H-Index: 23
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#1Sinéad T. Loughran (DCU: Dublin City University)H-Index: 8
#2Noeleen B. Loughran (DCU: Dublin City University)H-Index: 9
Last.Dermot Walls (DCU: Dublin City University)H-Index: 21
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#1Lin Li (POSTECH: Pohang University of Science and Technology)H-Index: 3
#2Dong Gyun Kang (POSTECH: Pohang University of Science and Technology)H-Index: 16
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Cited By5
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#1Shu-Chen Kan (NCHU: National Chung Hsing University)H-Index: 5
#2Chi-Ming Chen (NCHU: National Chung Hsing University)H-Index: 2
Last.Yung-Chuan Liu (NCHU: National Chung Hsing University)H-Index: 24
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#1Chia-Chi Lin (NCHU: National Chung Hsing University)H-Index: 5
#2Tzu-Tsen Liu (NCHU: National Chung Hsing University)H-Index: 2
Last.Yung-Chuan Liu (NCHU: National Chung Hsing University)H-Index: 24
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#1Siyavuya Ishmael Bulani (University of the Free State)H-Index: 1
#2Lucy N. Moleleki (University of Pretoria)H-Index: 12
Last.Ntsane Moleleki (CSIR: Council of Scientific and Industrial Research)H-Index: 2
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