Please suggest the available wet lab procedures by which i can find out the exact 'drug bound inhibited protein' of that bacteria.
One method is to select for resistant cells by growing a large amount of the bacteria on agar containing the drug at a concentration above the MIC. Then grow up several of the resistant colonies, isolate the genomic DNA, and sequence the genomes. Compare the genomes of the resistant colonies to the genome of the parent strain to see what has changed. Sometimes, there will be a mutation in the molecular target. Other times, there will be mutations elsewhere.
Another method is the pull-down experiment. A version of the antimicrobial compound is made that can be covalently attached to a resin to make an affinity resin. A cell extract is passed over the affinity resin, and also over a control resin that lacks the compound. The proteins that bound to the two resins are sequenced by mass spectrometry. Proteins that are found only on the affinity resin are candidates for the molecular target. Overexpress each of the candidates in the bacteria to see if any of them cause resistance to the compound, which is likely to occur if the target is overexpressed.
Thank you Adam B Shapiro sir, 2nd method seems to be more easier. Sir, will you please mention some of your papers (works using this procedure), so that i can get a clear idea about the procedure and all requirements (equipment) .
Also thanks to you Navid J. Ayon sir.
Here are a few references on the subject:
Article Biochemical Target Isolation for Novices: Affinity-Based Strategies
Article Proteomic methods for drug target discovery
Thank you sir. Im reading these articles. Best wishes sir Adam B Shapiro .
I read the articles. these provided many information which r very helpful.
Sir, what procedures do you follow to know the sequences of the proteins that r bound with immobilized compound (drug+resin) Adam B Shapiro , I mean amino acid sequences of the proteins e.g. MLRGTY..............
Mass spectrometry (LC-MS-MS) of a trypsin extract of the recovered proteins from the pull-down is used to sequence the peptides. The sequences are compared to the genome sequence of the organism, or a closely-related one, to identify the proteins. Each of the proteins so identified can be considered as a candidate target. Further experiments are needed to ascertain whether any of them is really the target.
One such experiment is prepare the purified protein and see whether the compound binds to it.
Another experiment is to overexpress the protein in the organism and see if the organism becomes less susceptible to growth inhibition by the compound.
Another experiment is the reduce the expression of the protein in the organism and see if the organism becomes more susceptible.
ohhh, These're impressive, Surely these will help me to construct my workflow. Thank you sir, a lot. One more favor, will you plz recommend some of your worked papers( resin bining to protein sequencing and validation), so that i can use the procedures and cite them.
Thank you
Isolation of resistant mutants and whole-genome sequencing, and biochemical assay for confirmation of mechanism:
Article Small-Molecule Inhibitors of Gram-Negative Lipoprotein Traff...
https://jb.asm.org/content/197/10/1726.long
Another, classical, method would be photoaffinity labelling. Some substances are natural photolabels (e.g., all nucleotides), others are modified with, say, an azido group. The substance also needs to be radioactive. Then you incubate the cytosol with your label, photolyse with UV-light, separate proteins from unbound label with a desalting column and do an SDS-PAGE followed by autoradiography. Identify the radioactive proteins, excise and identify. Note: Labelling with the radioactive compound should be preventable with excess non-radioacvtive compound, otherwise you are looking at non-specific binding.
Thank u sir Engelbert Buxbaum for this helpful information. Can u please refer any paper so that i can fully understand the workflow !!!
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