Expression, purification, and crystallization of neisserial outer membrane proteins

Area of Science:

• Microbiology
• Structural Biology
• Protein Chemistry

Background:

• Integral outer membrane proteins (OMPs) are crucial for Neisseria functions, including virulence and antigenicity.
• Studying Neisserial OMPs requires large quantities, posing challenges for direct isolation from bacterial cultures.
• Recombinant expression in E. coli followed by refolding is a common strategy for OMP purification.

Purpose of the Study:

• To describe an optimized method for the recombinant expression and purification of Neisseria outer membrane proteins (OMPs).
• To detail a protocol for the refolding and subsequent crystallization of the PorA porin from Neisseria.

Main Methods:

• Recombinant expression of OMPs in E. coli, leading to formation of inclusion bodies.
• Solubilization of inclusion bodies and refolding of the OMPs.
• Purification of refolded OMPs to milligram quantities.
• Crystallization trials for the refolded PorA porin.

Main Results:

• An optimized protocol for expressing and purifying Neisseria OMPs, exemplified by PorA, was established.
• The method yields milligram quantities of purified OMPs suitable for further structural and biophysical analyses.
• An approach for the crystallization of the PorA porin was successfully developed.

Conclusions:

• The described method provides a scalable and convenient approach for obtaining Neisseria OMPs for research.
• This technique facilitates structural, biophysical, and immunological studies of these important bacterial proteins.
• The developed crystallization method opens avenues for high-resolution structural determination of PorA.