Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4

• His6-tagged and untagged hGal4-CRD2 were produced in Escherichia coli.
• Recombinant proteins were purified by affinity chromatography and gel filtration.
• CD and fluorescence techniques were applied to asses correct folding and activity.
• Quality crystals were obtained by vapor-diffusion sitting drop method.
• The hGal4-CRD2 structure was solved by molecular replacement at 1.78 Å resolution.

Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.