Whole genome cloning and expression of Treponema pallidum open reading frames
EMSL Project ID
2291
Abstract
Treponema pallidum is a spirochete bacterium and is the causitive agent of syphilis. The complete genome sequence of T. pallidum is known. The organism has a genome size of 1.1 Mb and encodes 1031 open reading frames. In recent studies we have cloned each of the 1031 open reading frames into a plasmid that will direct protein expression in E. coli. Therefore, we are now in a position to express and purify each of the proteins encoded by the 1031 open reading frames of T. pallidum. A critical experiment is to determine what fraction of the 1031 proteins are actually expressed in E. coli cells and therefore available for purification. To determine the fraction of expressed proteins we propose to combine the E. coli strains into pools of 24 clones each and purify proteins from each pool. The purified proteins will then be digested with trypsin and analyzed by mass spectrometry. The identity of the proteins present in each pool can be determined from precise determination of the molecular weight of peptides. In this way it will be possible to rapidly determine what fraction of the 1031 T. pallidum open reading frames are expressed in E. coli. To accomplish these goals approximately 40 hours of use of an Ion Trap mass spectrometer is needed.
Project Details
Project type
Exploratory Research
Start Date
2001-08-01
End Date
2004-05-03
Status
Closed
Released Data Link
Team
Principal Investigator