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Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: an Application to a Protein Expressed as an Inclusion Body

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Title
Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: an Application to a Protein Expressed as an Inclusion Body
Authors
Kim, Hak Jun
Kang, Sung-Ho
Keywords
EPSP synthase; Expressed protein ligation; Herbicide; Intein
Issue Date
2007
Citation
Kim, Hak Jun, Kang, Sung-Ho. 2007. Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: an Application to a Protein Expressed as an Inclusion Body. 한국미생물생명공학회. 한국미생물생명공학회. 2007.06.29~.
Abstract
Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted to address various biological questions. EPL of the 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, was attempted. First, EPSP synthase was split into the N- and C-terminal fragments, based upon the cysteine scanning mutagenesis. Each domain was fused to the intein proteins. The N-terminal fragment (EPSPSN, 1-237) was purified from the intein-fused protein with a thioester tag exposed at its C-terminus. The C-terminal fragment (EPSPSCCYS, residues 238CYS-427) expressed as an inclusion body, was purified from a strain coexpressing the C-terminal fragment (Met-238CYS-427) and an additional methionine aminopeptidase (MAP) which enhances the removal of the initiation methionine from the C-terminal fragment. The purified fragments were mixed to initiate ligation in the presence of 2 % thiophenol. The ligation efficiency was ~85 %. The ligated full-length EPSP synthase was refolded and further purified using Mono Q anion exchange column. Circular dichroism (CD) and enzyme activity assays showed that the ligated full-length EPSP synthase has ~35 % specific activity compared to that of wild-type EPSP synthase.
URI
http://repository.kopri.re.kr/handle/201206/7704
Conference Name
한국미생물생명공학회
Conference Place
한국미생물생명공학회
Conference Date
2007.06.29~
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