Galectin-1 as a fusion partner for the production of soluble and folded human beta-1,4-galactosyltransferase-T7 in E. coli.
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 (beta4Gal-T7), in E. coli. The enzyme beta4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, beta4Gal-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-beta4Gal-T7 fusion protein, the unique protease cleavage site allows the protein beta4Gal-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 beta4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.
Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD, USA.
Marta
M
Pasek
Elizabeth
E
Boeggeman
Boopathy
B
Ramakrishnan
Pradman K
PK
Qasba
D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037483:Galectin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D014176:Protein Biosynthesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D037483:Galectin 1; C445499:LGALS1 protein, human; D011993:Recombinant Fusion Proteins; D005700:Galactosyltransferases; C063053:xylosylprotein 4-beta-galactosyltransferase