Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis.

Abstract

Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase.

• Authors: J S Oliveira, C A Pinto, L A Basso, D S Santos
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 22 Issue 3 Pg. 430-5 (Aug 2001) ISSN: 1046-5928 [Print] United States
• PMID: 11483005 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2001 Academic Press.

Chemical References

• Recombinant Proteins
• 5-enolpyruvoylshikimate-3-phosphate
• Shikimic Acid
• Phosphotransferases (Alcohol Group Acceptor)
• shikimate kinase

Topics

• Cloning, Molecular
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics)
• Mycobacterium tuberculosis (enzymology)
• Phosphotransferases (Alcohol Group Acceptor) (genetics, metabolism)
• Recombinant Proteins (analysis)
• Shikimic Acid (analogs & derivatives, metabolism)