E.coli  EXPRESSION,  PURIFICATION AND BINDING STUDIES OF GALECTIN-4

 

Veronika Suchá1, Gabriela Jeníková2, Vladimíra Marková2, Milan Fabry1, Petr Malý2,
Jiří Brynda1

 

1Department of Recombinant Expression and Structural Biology, Institute of molecular genetics, Flemingovo nám. 2, 166 37 Praha 6

2Department of Molecular Glycobiology, Institute of molecular genetics, Vídeňská 1083, 142 20 Praha 4

 

Galectins belong to a family of carbohydrate-binding proteins that share conserved amino acid sequences and affinity for ß-galactoside sugars.

Galectin-4 is a monomer of about 36 kDa containing two carbohydrate-binding domains (CDR1 and CRD2, 40% identical) within a single polypeptide chain [1]. This protein is expressed only in the alimentary tract, from the tongue to the large intestine. Strong expression of galectin-4 can be induced in cancers from other tissues including breast and liver. This distinct induction can make a valuable diagnostic marker and target for the development of inhibitory carbohydrate-based drugs.

Galectins may also bind intracellular non-carbohydrate ligands and have intracellular regulatory roles in processes such as RNA splicing, apoptosis, and, as suggested most recently, the cell cycle [2].

The exact function of galectin-4 has not been exactly found out yet.

 

Full-length mouse galectin-4 and its respective carbohydrate recognition domains CRD1 and CRD2 were expressed in E. coli and consecutively purified. Lactose-binding affinity of CDR1 was determined by fluorescence assay based on fluorescence quantum yield of two tryptophan residues (Trp 62, Trp 75) located on opposite borders of the binding site.

 

Specificity of recombinant galectin-4 and its respective carbohydrate recognition domains CRD1 and CRD2 labeled with FITC was determined using a glycan array consisting of synthetic and natural carbohydrates attached to microtiter plates. http://www.functionalglycomics.org/static/consortium/main.shtml

 

 

[1]  M.A. Gitt, C. Colnot, F. Poirier, K.J.Nani, S.H. Barondes, H. Leffler, J. Biol.Chem, 273 (1997),         2954-2960

 

[2]  M.H. Huflejt, H. Leffler, Glycoconjugate J., 20 (2004), 247-255