Cloning, expression and purification of recombinant GapA-1 The gapA-1 gene from MC58 was cloned into the expression vector pCRT7/NT-TOPO to facilitate the expression and subsequent purification of 6 × histidine-tagged recombinant GapA-1 (Figure 1a). This was used to generate RαGapA-1. Immunoblot analysis confirmed that RαGapA-1 and anti-pentahistidine antibodies both reacted to the purified recombinant GapA-1 (Figure 1b &1c). Figure 1 SDS-PAGE and immunoblot analysis of

recombinant GapA-1. SDS-PAGE analysis confirms the purity of the recombinant GapA-1 purified under denaturing Kinase Inhibitor Library conditions (a). Immunoblot analysis shows that recombinant GapA-1 is recognized by RαGapA-1 (b) and anti-pentahistidine antibodies (c). Construction of an N. meningitidis gapA-1 null mutant strain To examine the roles of GapA-1 in the meningococcus, a gapA-1 knockout derivative of N. meningitidis MC58 was generated. Immunoblotting using RαGapA-1 showed that GapA-1 could be detected in whole cell lysates of wild-type but not MC58ΔgapA-1 (Figure 2, lanes 1 & 2) confirming that GapA-1 was expressed under the conditions used and that expression had been abolished in the mutant. This analysis further confirmed that the Selleckchem Z-IETD-FMK RαGapA-1 sera did not recognize GapA-2 (37-kDa) under the conditions used. To further confirm that the immuno-reactive protein was GapA-1, a wild-type copy of

gapA-1 was introduced in trans into MC58ΔgapA-1 using plasmid pSAT-14 (Table 1). Introduction of gapA-1

at an ectopic site restored GapA-1 expression (Figure 2, lane 3). Further immunoblot analyses using old a panel of 14 N. meningitidis strains (Additional file 1) including representatives of differing serogroups and MLST-types showed that GapA-1 expression was conserved across all strains (data not shown). Expression was also conserved in N. gonorrhoeae FA1090 (data not shown). These data complement in silico predictions that GapA-1 is universally AZD0156 order present and suggests that GapA-1 is constitutively-expressed across pathogenic Neisseria species. Figure 2 Immunoblot analysis of whole cell proteins from N. meningitidis using RαGapA-1. Analysis of MC58 wild-type, ΔgapA-1 mutant derivative and complemented mutant reveals the absence of GapA-1 in the ΔgapA-1 mutant preparation. Similar analysis shows the abolition of GapA-1 expression in the MC58ΔsiaD ΔgapA-1 mutant compared to the parental MC58ΔsiaD strain. Meningococcal GapA-1 is only surface-accessible to antibodies in the absence of capsule Grifantini et al showed using flow cytometry that GapA-1 was accessible to specific antibodies on the surface of meningococci [27]. However, the methodology used involved pre-treatment of the cells with 70% ethanol to permeabilize the capsule, making it unclear whether GapA-1 was accessible to antibodies in encapsulated bacteria.