Long story short, I need to clone several enzymes of a metabolic pathway I'm interested in. I'd like to test my molecule that I believe inhibits the last enzyme of the pathway I'm interested in. I need the two enzymes before the last enzyme because the molecule I made needs to be metabolized by them in theory in order to make the active form of the molecule. I have no experience with cloning proteins, but if were to attempt this, what kind of time frame am I looking at? How can I be sure I'll get the active forms of my enzyme as well after cloning?
Cloning several metabolic enzymes to screen for inhibition, how long?
Posted 07 May 2015 - 01:13 PM
First off... you clone DNA not protein... However, we understand what you mean. You can't be sure that you will get active forms until you try it, and this usually depends on what organism you plan to express in. It is probably best to stick with expressing in the organism that your enzymes come from (or closely related e.g.for a human enzyme, you could express in mouse; for bacteria, use a bacterial system, etc.). You might also want to work in a system that is deficient in these enzymes in the first place, so that you can be certain that the results you see are due to the enzymes themselves. For mammalian systems you can get rabbit reticulocyte lysate that can do this. Bacterial and yeast have gene knockouts for a lot of things.
Depending on the methods you use, cloning can take as little as a week, up to about 6 months (worst case). We need a bit of information about how you plan to go about this. Do you already have the genes in a form that can be amplified (RNA/cDNA, or on another plasmid)? Or do you need to start from scratch to get the RNA? Do you know the sequences you are expecting?
If you know the sequence, but can't get it elsewhere, you can order them from a number of companies (cost about $0.2 per base pair) and they usually come in a "ready to clone" format in a basic plasmid such as pUC. If you know the vector you want to express them off, you can design the sequences with the desired restriction sites on each end, so that it is a cut and ligate job.
If you have them on another plasmid or cDNA and can amplify them, then it is relatively easy to PCR (either with desired restriction sites or use TA cloning) and then insert into another vector. Another thing to think about is seeing if the gene is available in a plasmid from places like addgene. Plasmids can either be used as a template for PCR or for subcloning, or, if you get them in the right plasmid, for expression directly.