title: Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.
abstract: 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.
authors.affiliation: Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café, s/n, 14040-903 Ribeirão Preto-SP, Brazil.
authors.forename: Joane K
authors.initials: JK
authors.lastname: Rustiguel
authors.affiliation: Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de São Paulo, Av. João Dagnone, 1100, 13563-120 São Carlos-SP, Brazil.
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Kumagai
authors.affiliation: Laboratório de Glicoimunologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café, s/n, 14040-903 Ribeirão Preto-SP, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Dias-Baruffi
authors.affiliation: Laboratório de Biofísica Molecular, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeirão Preto-SP, Brazil.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Costa-Filho
authors.affiliation: Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café, s/n, 14040-903 Ribeirão Preto-SP, Brazil. Electronic address: cristy@fcfrp.usp.br.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Nonato
mesh_terms: D001665:Binding Sites; D001703:Biophysics; D002241:Carbohydrates / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D037541:Galectin 4 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D037541:Galectin 4; D011993:Recombinant Fusion Proteins
keywords: Carbohydrate recognition domain; Circular dichroism; Crystal structure; Fluorescence; Human galectin-4; X-ray diffraction