Expression, Purification, and Crystallization of Neisserial Outer Membrane Proteins
Integral outer membrane proteins (OMPs) play key roles in solute transport, adhesion, and other processes. In Neisseria, they can also function as major protective antigens. Structural, biophysical, and immunological studies of Neisserial OMPs require the
- PDF / 994,621 Bytes
- 16 Pages / 504.57 x 720 pts Page_size
- 51 Downloads / 203 Views
Introduction Structural studies on porins and other integral outer membrane proteins (OMPs) have established that they generally adopt a transmembrane B-barrel structure, with an even but varying number of B-strands. This creates a highly stable and versatile structure, which can be adapted for use as a pore-forming protein, an energized transporter or an adhesin for recognition of host cell surface receptors (1). In comparison with their transmembrane helix-containing counterparts, OMPs are generally more stable in the detergentsolubilized state and their relative abundance in the Protein Data Bank suggests that they are more tractable to structure determination, traditionally by X-ray crystallography and, more recently, by solution state NMR. Investigators may also wish to isolate specific OMPs
Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_6, © Springer Science+Business Media, LLC 2012
91
92
M. Saleem et al.
for immunological purposes, particularly as vaccine components. The application of all these methods requires that the protein is isolated in high yield, and in its native state. A method which has been widely adopted for OMP isolation is to direct expression into inclusion bodies (IBs), resolubilize (e.g. using urea or guanidine HCl) and refold, by addition of detergent and simultaneous removal of chaotrope. This general method, with some modifications, has been reported for many of the main integral OMPs from Neisseria, including PorA (2), PorB (3), FetA (4), NspA (5), OpcA (6), and the opacity proteins (7). In some cases, crystal structures have been reported (3, 5, 6), providing strong evidence that the correct native state was obtained from refolding. Our experience has been that particular attention needs to be paid to the conditions for refolding, but that there may be several ways to achieve optimally refolded protein in high yield. Thus, although there are a variety of methods reported in the literature, it is possible to give some general guidance in devising a refolding protocol. Here, we have taken the PorA porin as an example; its importance as a meningococcal vaccine component is well established (8), and the methods employed can be generalized to other OMPs relatively easily. We also show how the purified OMP can then be fed into crystallization trials, and give an example of crystals obtained by this approach.
2. Materials 2.1. Expression of the Neisseria Outer Membrane Protein, PorA, as Inclusion Bodies (IBs)
1. PorA plasmid expression vector (see Notes 1 and 2). 2. T7 Express Competent E. coli (New England Biolabs). 3. Luria Bertani (LB) agar and LB liquid medium broth (Fisher). 4. 2xYT liquid medium broth: add 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl to 1 L of deionized H2O. Alternatively, purchase 2xYT broth powder (Fisher). 5. Kanamycin (30 mg/mL stock concentration, stored at −20°C). 6. Isopropyl B-D-1-thiogalactopyranoside, IPTG (1 M stock). 7. Baffled Erlenmey
Data Loading...
