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Title: Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

Abstract

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification ofmore » galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Authors:
 [1]; ;  [1]
  1. Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States)
Publication Date:
OSTI Identifier:
22202464
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 394; Journal Issue: 3; Other Information: Copyright (c) 2010 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ENZYMES; ESCHERICHIA COLI; GALACTOSE; LACTOSE; MALTOSE; XYLOSE

Citation Formats

Pasek, Marta, Boeggeman, Elizabeth, Ramakrishnan, Boopathy, Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170, and Qasba, Pradman K., E-mail: qasba@helix.nih.gov. Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli. United States: N. p., 2010. Web. doi:10.1016/J.BBRC.2010.03.051.
Pasek, Marta, Boeggeman, Elizabeth, Ramakrishnan, Boopathy, Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170, & Qasba, Pradman K., E-mail: qasba@helix.nih.gov. Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli. United States. https://doi.org/10.1016/J.BBRC.2010.03.051
Pasek, Marta, Boeggeman, Elizabeth, Ramakrishnan, Boopathy, Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170, and Qasba, Pradman K., E-mail: qasba@helix.nih.gov. Fri . "Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli". United States. https://doi.org/10.1016/J.BBRC.2010.03.051.
@article{osti_22202464,
title = {Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli},
author = {Pasek, Marta and Boeggeman, Elizabeth and Ramakrishnan, Boopathy and Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 and Qasba, Pradman K., E-mail: qasba@helix.nih.gov},
abstractNote = {The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.},
doi = {10.1016/J.BBRC.2010.03.051},
url = {https://www.osti.gov/biblio/22202464}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 3,
volume = 394,
place = {United States},
year = {2010},
month = {4}
}