The following work was accomplished in 2009: Expression, Purification and Complexation of Bacterial and Human Proteins An expression system for TbpA has been developed and is capable of producing 0.2-0.4 mg/L of purified protein. A large scale preparation (27L) of E. coli transformed with a plasmid containing the gene for TbpA as well as an N-terminal His-Tag and a cleavage site are grown and induced with low level IPTG overnight and then harvested. We recently improved expression yields through targeted mutagenesis. We also introduced additional methionine residues for phasing of TbpA crystals. Purified TbpA forms crystals in 96 well plates which are being optimized. An expression system for TbpB has also been developed and is capable of producing 7-8 mg/L of purified protein. A medium scale preparation (4L) of E. coli transformed with a plasmid containing the gene for TbpB (a soluble construct lacking the N-terminal membrane anchor) as well as an N-terminal His-Tag and a cleavage site are grown and induced with high level IPTG for a few hours and then harvested. The cells are then lysed and membranes are separated via ultracentrifugation, and the soluble fraction is run over a Nickel-NTA column. The Nickel eluate is then further purified and can be bound to purified commercially available iron-bound human serum transferrin. Recently, we made the first in vitro complex of TbpA-TbpB bound to human transferring (hTf) and this complex will be characterized by single particle electron microscopy. We also have crystallized TbpA in complex with hTf and have obtained a partial molecular replacement solution for the crystal structure using the coordinates of hTf which we solved in 2007.
We aim to provide structures of TbpA alone, TbpA-hTf, and TbpA-TbpB-hTf in the coming year.
| Noinaj, Nicholas; Cornelissen, Cynthia Nau; Buchanan, Susan K (2013) Structural insight into the lactoferrin receptors from pathogenic Neisseria. J Struct Biol 184:83-92 |
| Noinaj, Nicholas; Buchanan, Susan K; Cornelissen, Cynthia Nau (2012) The transferrin-iron import system from pathogenic Neisseria species. Mol Microbiol 86:246-57 |
| Noinaj, Nicholas; Easley, Nicole C; Oke, Muse et al. (2012) Structural basis for iron piracy by pathogenic Neisseria. Nature 483:53-8 |
| Noinaj, Nicholas; Fairman, James W; Buchanan, Susan K (2011) The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex. J Mol Biol 407:248-60 |
| Noinaj, Nicholas; Guillier, Maude; Barnard, Travis J et al. (2010) TonB-dependent transporters: regulation, structure, and function. Annu Rev Microbiol 64:43-60 |
| Udho, Eshwar; Jakes, Karen S; Buchanan, Susan K et al. (2009) Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes. Proc Natl Acad Sci U S A 106:21990-5 |
