Preeti Thakur- University of Massachusetts Chan Medical School
Preeti Thakur
- University of Massachusetts Chan Medical School
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21
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Publications (21)
Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis path...
Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylat...
Abstract The preprotein translocase, YidC is an envelope protein which controls respiratory metabolism in Mycobacterium tuberculosis. Previously, we have established that depletion of yidC is deleterious for both extra- and intracellular proliferation of M. tuberculosis; however, it remains unclear how YidC expression is regulated under different g...
The YidC–Oxa1–Alb3 preprotein translocases play a vital role in membrane insertion of proteins in eukaryotes and bacteria. In a recent study we observed that Rv3921c, which encodes putative YidC translocase in Mycobacterium tuberculosis (Mtb), is essential for in vitro growth of bacteria. However, the exact function of this particular protein remai...
Recombination-based tools for introducing targeted genomic mutations in Mycobacterium tuberculosis are not efficient due to higher rate of illegitimate recombination compared with homologous DNA exchange. Moreover, involvement of multiple steps and specialized reagents make these tools cost ineffective. Here we introduce a novel clustered regularly...
Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of...
Phylogenetic tree analysis of microbial EngA proteins. An unrooted Phylogenetic tree was constructed from an alignment of MSMEG_3738 with the orthologous sequences as listed in Table 1, by using neighbor joining method [27]. The numbers in the parenthesis next to each organism represent the calculated distance values that reflect the degree of dive...
Alignment of MSMEG_3738 with EngA protein sequences of other mycobacterial species. Homologues of MSMEG_3738 were identified by blastp homology searches in different mycobacterial species that include M. abscessus (Mab), M. avium subsp. paratuberculosis K-10 (Map K-10), M. avium subsp. paratuberculosis S397 (Map S-397), M. gilvum (Mgi), M. intracel...
Alignment of MSMEG_3738 with Der protein sequence of T. maritima. MSMEG_3738 was aligned with Der protein sequence of T. maritima by using AlignX program of Vector NTI software as described in materials and methods section. The number in parentheses before each sequence represents the position of amino acid residue of EngA protein sequence in the a...
Comparative analysis of engA locus organizations in different mycobacterial species. Organizations of genes in engA locus of different mycobacterial species were analyzed by “genome region comparison” tool of CMR database (http://cmr.jcvi.org). Analysis of engA locus in different mycobacterial species indicates a conserved occurrence of genes prece...
In vitro GTPase activity analysis of EngAMS. A) GTPase assay was performed as described in the text with (GTP+EngA) or without (GTP only) 1 µM EngAMS in the reaction mixtures containing different concentrations of GTP. Shown is the bar graph plot using values of the rate of GTP hydrolysis (µM Pi released per hour) and concentrations of GTP (µM), re...
EngAMS exhibits interaction with ribosome in vivo. A) The E. coli BL21 (DE3) cells overexpressing EngAMS were lysed in RNase-free environment by repeated freeze-thaw cycles and fractionated in apo form (lacking nucleotide) on 10–45% sucrose gradient prepared in low salt buffer (containing 30 mM NH4Cl), by ultra-centrifugation (using Beckman SW28 ro...
Homology modeling predicts interactions of GD-1 and GD-2 with KH domain of EngAMS. A) Homology model prediction of EngAMS using structure of Der protein of T. maritima proposes interaction of C-terminal KH domain with both the G-domains. Specific amino acid residues involved in D1-KH interaction are part of G3 motif (B), whereas those participating...
Sequences exhibiting significant alignments with MSMEG_3738. Homologues of EngAMS were obtained by blastp search as described in the materials and methods section. The table shows a list of top 100 organisms that contain EngA protein exhibiting close homology with EngAMS and used in the Phylogenetic analysis. The accession number of each of the Eng...
Both the G-domains of EngAMS are required for binding with GDP. Nucleotide binding was assayed by recording fluorescent intensities at 460 nm (λex 355 nm) upon incubating wild-type (WT) and point mutant derivatives (G4_D1 and G4_D2, respectively) of EngAMS protein with fluorescent mant-nucleotide (mant-GDP), as described in the materials and method...
List of bacterial strains, primers and plasmid constructs used in the study. Table shows the complete list of bacterial strains, primers and plasmid constructs used in this study, as mentioned in the text. The underlined primer sequences represent respective restriction endonuclease recognition sites.
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Questions
Questions (2)
I am trying to modify a retroviral vector PMIG II which has 5' LTR and 3' LTR used as promoters. The 5'LTR is followed by multiple cloning site (MCS). I am looking to clone my gene of interest under granzyme B mouse promoter at MCS site. Do I need to remove 5'LTR and 3'LTR in the vector in order to do that? Can 5'LTR drive my granzyme B Promoter?. The vector would be ultimately used to transduce mouse cell line.
Thanks in advance for the feedback.
I am trying to purify a N-terminally GST tagged protein which is very well expressed in E.coli membrane. I was successful in extracting it out from the membrane using sarcosyl/detergents but the protein is somehow having low binding onto the column, most of the part seen in flow through. However I was able to purify soluble GST with the same buffers which I used for the protein of interest. What could be the reason for protein low binding onto column?
