W0092

Genomics Approach to Expression of Membrane Proteins From Mycobacterium Tuberculosis. Y. Peskova, K. DiGiandomenico, F. Gao, Y. Hua, A. Korepanova. T. Cross, and R. Nakamoto., Mol. Physiology & Biol. Physics, Univ. of Virginia, 1300 Jefferson Park Ave., Charlottesville, VA 22908 USA.

We are exploring the membrane protein expression space by testing high yield expression of all the open reading frames (ORFs) of the predicted membrane proteins of Mycobacterium tuberculosis. To do this we are creating an expression library of all of the open reading frames (ORFs) for predicted membrane proteins using the TOPO and GATEWAY cloning systems (Invitrogen), which are amenable to high throughput approaches. Each PCR-amplified ORF is inserted into four different E. coli expression vectors: two with amino terminal tags and two with carboxyl terminal tags. The other end of the protein is left with native sequence. Polyhistidine or Strep II tags are used with the strong T7 promoter or the weaker tet promoter. Up to now, we have tested more than 120 ORFs in different strains of E. coli and expression is observed with more than 80. Several tendencies were observed: (1) the most successful strain for expression of membrane proteins is the BL21 derivative, C43. (2) Proteins that express at high levels usually have amino terminal tags. (3) Higher levels of expression were always obtained with the T7 promoter. (4) There appears to be little correlation between the level of expression and the number of predicted membrane spanning helices or relative content of hydrophilic vs. hydrophobic domains. This work was supported by PHS grant P01-GM64676.