|
Gene expression in prokaryotic system |
| Principal Investigator : Lalit C Garg
Project Associates
/ Assistants Ph D Students Collaborators 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. Cloning,
expression, purification and characterization of proteins from glycolytic
pathways 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. 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. The
present proposal, therefore, aims to clone, express and characterize the
enzymes of glycolytic pathway (with the main emphasis to the one directly
catalyzing the energy producing reactions) of M.tuberculosis, as glycolysis is
a primary energy producing pathway and is of great importance in all the
organisms. For this purpose, genes encoding the glycolytic pathway enzymes of
M.tuberculosis (enolase, glyceraldehydes–3-phosphate dehydrogenase, putative
phosphoglycero mutase1, phosphoglycerate kinase, phosphoglycerate mutase,
fructose bisphosphate dehydrogenase, aldolase, glucose 6 phosphate isomerase,
putative phosphoglycerate mutase2, pyruvate kinase, phosphofructokinase A,
phosphofructokinase B, triosephosphate isomerase) were PCR amplified from
M.tuberculosis genomic DNA using gene specific primers on the basis of
nucleotide sequence of M.tuberculosis genome available in the GenBank. PCR
amplified fragments were gel purified and cloned in pGEM-Teasy vector.
Recombinant clones were selected by restriction enzyme analysis. Gene
fragments coding for different enzymes of M. tuberculosis glycolytic pathways
were released from pGEM-Teasy clones by suitable restriction enzymes and
cloned into pET22(b)+ vector digested with the same enzymes. The constructs
thus obtained had the gene under the control of T7 promoter and 6X His tag at
the 3’ end of the gene. BL21
cells harbouring the recombinant plasmids were grown in LB in the presence of
ampicillin (100 mg/ml). The cultures were induced with 1 mM IPTG and were
further grown for ~ 8 h. Expression of different genes in total cell extracts
from both uninduced and induced cultures was analysed by 15% SDS-PAGE. A
predominant band corresponding to the expected size of each protein was
observed in respective induced cultures. These data suggest that the
expression of the recombinant protein with histidine tag was efficiently
directed by T7 promoter. Low level expression of these genes was observed in
uninduced cultures. However, the expression of putative phosphoglycerate
mutase 2 gene could not be achieved. His-tagged
recombinant proteins accumulating in the form of inclusion bodies were
solubilized in 6M guanidine hydrochloride and purified by Ni-NTA
chromatography. A single band on SDS-PAG confirmed the purity of the protein.
At shake flask level, the yield of pure recombinant proteins was about 15-20
mg per liter of culture. Phosphoglucose
isomerase with 6Xhis tag, with an apparent subunit mass of 59.97 kDa, was
expressed as inclusion bodies. The enzyme was purified and refolded
successfully by one step immobilized metal ion affinity chromatography. The
purified enzyme was analysed for its Km, Ki, pH optima. The enzyme exhibited a
bell shaped curve with maximum activity at pH 7.6-8.5. The
recombinant protein had a Km of 2.7 + 0.03 mM for fructose-6-phosphate. Ki for
the competitive inhibitor 6-phosphogluconate was 0.7 mM. The recombinant
protein remained stable and active for several weeks even when stored at room
temperature. Thermal stability of M. tuberculosis recombinant PGI was
determined by assaying for enzyme activity after incubation at temperatures
between 4-80°C. The enzyme retains its activity upto 55°C. Presence of 2 M
urea in the assay buffer resulted in 85% loss of activity. Mn2+ and Ca2+ (5
and 20 mM, respectively) increased the activity of the recombinant enzyme by
50%. Triosephosphate
isomerase (TPI) was expressed as a soluble protein and further
characterization of the recombinant enzyme is in progress. |