Refolding, purification and crystallization of the FrpB outer membrane iron transporter fromNeisseria meningitidis

Abstract

FrpB is an integral outer membrane protein from the human pathogenNeisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed inEscherichia coliinto inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space groupC2, with unit-cell parametersa= 176.5,b= 79.4,c= 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space groupP212121, with unit-cell parametersa= 85.3,b = 104.6,c= 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit.