title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P2₁ and P2₁2₁2₁, respectively. The structure was solved by molecular replacement and structure refinement is now in progress.
authors.affiliation: Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
authors.forename: Revathi
authors.initials: R
authors.lastname: Nagarajan
authors.affiliation: Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
authors.forename: Karthe
authors.initials: K
authors.lastname: Ponnuraj
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013292:Streptococcus agalactiae / Q000737:chemistry* / Q000201:enzymology; D040681:Structural Homology, Protein
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: glyceraldehyde-3-phosphate dehydrogenase; group B streptococcus; moonlighting; plasminogen; surface adhesin