I have amplified and cloned a gene from bacteria. It has good expression in pET28-Rosetta. Moreover, it is soluble and active towards substrate.
Now, lets assume I have to check its catalytic domain (active site) and need to do mutation in gene sequence. Anyone can suggest me some of the more easy, low cost and reliable methods to check enzyme active site by doing mutation? Thanks in advance.
Dear Themina
first of all you need to find out the residues part of the catalitic site and generally you can do it by computing the sequence of your protein with the ones of enzymes characterized.
try to run smart (http://smart.embl-heidelberg.de/)
once you have determined the site and the function of the enzyme, you have to produce single point mutants to inactivate it by replacing one ore more of the catalitic residues and test it again in activity assay or substrate binding assay depending from the function of the enzyme.
as tool to ready perform mutagenesis i suggest to yuo PIPE cloning ( you can find more information about it on my blog: https://proteocool.blogspot.com/?m=1
at page 1: Cloning
regarding binding assay, if your substrate is a small molecule you can try dsf (differential scanning fluoeimetry) which is simple and cheap of in your lab there is a Real time pcr available. otherwise you need to perform specific activity assay.
good luck
Manuele
Dear Dr. Manuele! Thank you so much for your detailed and value added answer. May I have your email id to contact you?
Hello Tehmina hope you are fine. If you have sequence of your gene it is quite easy. you can use embl as suggested by Manuele Martinelli . But before starting experiment, I would suggest to double check the catalytic residues the best way for me is homology modeling and check if related structures are already submitted to pdb database compare the catalytic residues and you can also use the pymol etc to visualize the catalytic residues of your protein and their interaction. You can select the catalytic site residues and go for site directed mutagensis. For my experiments, I designed the primers to change target residues and used Quickchange mutagensis kit and got good results. Good luck with your experiment.
Thank you so much Uzma Hameed dear. I am fine and hope so that you are too. Thanks a lot for your detailed answer and sharing valuable suggestions.
Assalam-o-Alaikum, hope you are fine. if you have sequence, then binding pocket/active site needs to be identified first, by using various tools available online like Metapocket, fpocket, Patchdock etc. You can use UCSF Chimera to visualize protein structure and its detailed characteristics. You can confirm identified active site pocket using AutoDock tools (ADT). You can prepare your protein and ligand in pdb format. After having finished these steps you can run ADT, select protein pdb file, add hydrogen, minimize the energy of ligand first then select ligand pdb file and set-up covalent map and write pdb file for protein-ligand complex formed and check attachment position of ligand in active site pocket. You can choose one or more active site residues whose absence impart drastic effects on ligand binding or catalysis (based on characteristics of structure) and induce mutation by over-lap extension PCR to inactivate the protein and test the activity of mutant protein by enzyme assay.
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