Expressed protein ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase: An application to a protein expressed as an inclusion body SCIE SCOPUS KCI

Cited 2 time in WEB OF SCIENCE Cited 2 time in Scopus
Title
Expressed protein ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase: An application to a protein expressed as an inclusion body
Author(s)
Kim, Hak Jun; Shin, Hee Jae; Kim, Hyun Woo; Kang, Sung-Ho; Kim, Young Tae
Publication Year
2007-12-20
Abstract
Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its C-terminal fragment (EPSPSC, residues Mee(237)-238(CYS)-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing similar to 1 mM of EPSPSN-thioester with similar to 2 mM of EPSPSCCYS (residues 238(CYS)-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was similar to 85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and similar to 115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.
ISSN
0253-2964
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/4605
Bibliographic Citation
BULLETIN OF THE KOREAN CHEMICAL SOCIETY, v.28, no.12, pp.2303 - 2309, 2007
Publisher
KOREAN CHEMICAL SOC
Subject
YEAST PHOSPHOGLYCERATE KINASE; SITE-DIRECTED MUTAGENESIS; CHEMICAL LIGATION; ESCHERICHIA-COLI; 3-PHOSPHATE SYNTHASE; TERMINAL DOMAIN; HERBICIDE GLYPHOSATE; UNPROTECTED PEPTIDES; RECOMBINANT PROTEINS; SEMISYNTHESIS
Keywords
expressed protein ligation; intein; 5-Enolpyruvylshikimate-3-phosphate synthase; methionine aminopeptidase
Type
Article
Language
English
Document Type
Article
Publisher
KOREAN CHEMICAL SOC
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