Membrane Protein Expression in Mycobacterium Tuberculosis

1. Membrane Protein Expression in Mycobacterium Tuberculosis Jason Crowe, Clarkson University April Jue, University of North Carolina at Chapel Hill

2. Presentation Overview • Introduction to Membrane Protein Pilot Project • Overview of methods used • Results from experiments • Discussion of implications of project

3. Membrane Protein Pilot Project • Goals: observe, classify, and analyze membrane proteins of Mycobacterium tuberculosis • Target gene is selected, cloned, and the protein it codes for is expressed • Allows for analysis of protein through NMR, Mass spectroscopy, and X-ray diffraction

4. Membrane Proteins • Proteins located within cellular membrane • Needed for transporting nutrients, hormones, and other signals in and out of a cell • Control membrane potentials, which are vital to cellular processes • Structures and functions are difficult to determine, and thus mostly unknown

5. Plasmid Purification • This is done to separate the plasmid from the rest of the cellular material • Buffers are added to break apart the cellular membrane • Plasmids remain in solution while cellular material is centrifuged out • Plasmids eluted out of solution and run on agarose gel to verify presence of DNA plasmid

6. Protein Expression • Cell expresses target protein • Cells are grown in LB media, and expression is induced with IPTG • Induction causes cell to produce target protein almost exclusively • Cells are broken apart by sonication and soluble and insoluble fractions separated • Fractions run on SDS-page gel to determine presence of protein

7. Protein Purification • Purifies target protein using affinity binding or ion-exchange column • Ni+ Affinity binding column binds 6-histidine residue tag on protein • Ion-exchange column uses charge of protein to bind or pass through column

8. SDS-PAGE Protein gel Used to check protein expression and protein purification AGAROSE DNA gel Used to verify presence of DNA plasmid and plasmid insert Electrophoresis Gels

9. Protein Expression Results

10. DNA Agarose Gel

11. After Ni+ affinity binding After ion exchange Protein Purification Results

12. Protein for gene Rv2169c • Membrane protein composed of 134 amino acids • Molecular weight of 14556.7 Daltons • Isoelectric point at 11.7109 • This protein is as yet of unknown structure and function • TMHMM analysis shows 2 probable transmembrane regions

13. TMHMM analysis

14. Implications of Pilot Project • TB is #1 cause of infectious mortality in the world • Multi-drug resistance is a major concern, so new methods of fighting the disease are vital • Membrane proteins are the target for new treatments as they control many vital cellular activities

15. Difficulties with Expression • Membrane proteins are hydrophobic, and cells do not like to produce them • They require a lipid environment • Foreign proteins may not express well in expression systems • Membrane proteins tend to aggregate within cell and form inclusion bodies

16. Genomics of Membrane Proteins • 33% of a genome usually codes for membrane proteins, yet less than 1% of the proteins in the Protein Data Bank are membrane proteins • By determining structures of 50 membrane proteins, the Protein Data Bank’s collection of membrane protein structures will be tripled • Structure usually implicates function within membrane proteins

17. Thanks to: • Dr. Philip Gao, Mentor • Dr. Alla Korepanova • Yuanzi Hua, Lab Technician • The entire lab staff • Dr. Jack Crow, Director of the NHMFL • Pat Dixon, Gina Hickey, Paula Crone, and the rest of the CIRL staff