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Gene expression in prokaryotic system |
| Principal Investigator : Lalit C Garg
Project
Associates/Assistants
Ph
D Students
Colloborators The
goal of this project is to clone and express genes of biomedical importance,
and to understand the molecular mechanisms involved in the regulation of gene
expression. The main
objective of this study is to clone, express and characterize the enzymes of
glycolytic pathway of M.tuberculosis and to identify the structural and
functional differences between the enzymes from the pathogen and the host for
designing new selective inhibitors specific to M.tuberculosis enzymes. Expression,
purification and characterization of triosephosphate isomerase of M.tuberculosis Mycobacterium tuberculosis can metabolize a variety of carbohydrates and sequence analysis of M.tuberculosis has revealed that it possesses enzymes that can accomplish a variety of metabolic pathways. Glycolysis is a primary energy producing pathway and is of great importance in all the organisms. Identification of the differences between the enzymes from pathogen and the host is important as it provides opportunities for designing new selective inhibitors specific to M.tuberculosis enzymes. In the present study, our aim is to express and characterize triosephosphate isomerase (TPI). E.coli cells harboring the plasmid containing the TPI gene were induced. High level expression of triosephosphate isomerase with 6Xhis tag was observed as soluble protein in cytosolic fraction of induced cells. The recombinant TPI was purified to homogeneity by single step Ni-NTA affinity chromatography. At the shake flask level, the purified recombinant TPI was obtained with a yield of 10-12 mg/l of culture. Authenticity of the purified protein was confirmed by N-terminal sequencing. The molecular mass was determined as 28.213 kDa by mass spectrophotometry which matched with the expected molecular mass of the recombinant enzyme. The purified recombinant enzyme was further analyzed by CD spectra. The activity of the purified enzyme was measured at different pH under the standard assay system where the enzyme converts glyceraldehydes-3-phosphate to dihydroxy acetone phosphate which is reduced by the auxiliary enzyme a-glycerophosphate dehydrogenase and the oxidation of b-NADH is measured spectrophotometrically at 340 nm. The enzyme was active between pH 5.0 to 8.6, with maximum activity at pH 7.0. The purified enzyme was thermostable up to 55°C and showed complete loss of activity at 60°C. The purified enzyme did not show any requirement for cations as cofactors as the presence or absence of different monovalent or divalent cations had no effect on the activity. However, inhibition of the enzyme activity was observed at a very high cationic concentration. The Km of the recombinant enzyme for glyceraldehydes-3-phosphate as substrate was determined to be 2.45±0.03 mM. It
has been reported that the addition of a tag at the N- or C- termini of the
protein sometimes may change the functional activity of the protein. In order
to determine the effect of 6×his tag on TPI activity, TPI was cloned and
expressed without the 6×his tag. Like the 6×his tagged TPI, expression of
TPI without the 6×his tag was also obtained in cytosolic fraction as soluble
protein. Purification of the recombinant TPI without 6×his tag is underway. |