Molecular Cloning, Expression and Purification of Fructose-1, 6-bisphosphate Aldolase of Neisseria meningitidis-MC58

Abstract

Neisseria meningitidis is an exclusively human nasopharyngeal commensal, which remains a leading cause of lifethreatening meningitis and septicemia. Fructose-1, 6-bisphosphate aldolase (FBA) is a cytoplasmic glycolytic enzyme, which despite lacking identifiable secretion signals have also been shown to be transported to the surface of numerous bacteria and shown to possess diverse, non-glycolytic biological functions. In silico analysis of FBA sequences from N. meningitidis and N. gonorrhoeae shows a high degree of sequence conservation at the amino acid level, but only low-level (ca. 30%) homology to the FBA homologue from S. pneumoniae. The gene encoding FBA from N. meningitidis serogroup B strain MC58 was cloned in two different vectors carrying an N-terminal and C-terminal His-tags and expressed in host E. Coli. Attempts to express and purify the recombinant protein with an N-terminal fusion tag yielded mostly insoluble and inactive protein. However, the ca. 38-kDa recombinant protein corresponding to FBA was affinity-purified under denaturing conditions with N-terminal fusion tag and non-denaturing conditions with C-terminal fusion tag to near homogeneity and used to raise polyclonal monospecific rabbit serum to facilitate subsequent characterization of the enzyme. The molecular mass of the recombinant FBA was in agreement with the mass calculated on the basis of its gene sequence.