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.

Keywords: glyceraldehyde-3-phosphate dehydrogenase; group B streptococcus; moonlighting; plasminogen; surface adhesin.

Figure 1

12.5% SDS–PAGE of purified GAPDH. Lane 1, molecular-weight marker (labelled in kDa); lane 2, final purified rGAPDH at a concentration of 12 mg ml⁻¹.

Figure 2

Crystals of rGAPDH. (a) Needle-like crystals of approximately 0.03 × 0.05 × 1.0 mm in size obtained in 40%(w/v) PEG 3350, 0.1 M Tris–HCl pH 9.0. (b) Crystals obtained in 40%(w/v) PEG 4000, 0.1 M Tris–HCl pH 9.0. The rod-like crystals shown are about 0.1 × 0.1 × 0.8 mm in size.