Enzyme protocols

[Mark Ian]

Hello,

 

As you've might have noticed most enzymes on the market are ridiculously expensive compared to production costs. What I have heard from people I've worked with is that it semi legal to isolate enzymes (excluding high fidelity versions) if it is used for non commercial experiments. Besides I have never heard of a company going after a single person for producing enzymes, they concentrate on Universities and Institutes.

With that said I wanted to ask if you guys have any good protocols for isolating polymerases, ligases, Dna Ladders, restriction endonucleases or any other enzyme for the home laboratory.

 

thanks in advance

[CharonY]

In the olden days all the enzymes needed for cloning experiments were isolated by hand and not bought. It is not only semi-legal, but sometimes standard practice (also note that DNA ladders are, of course, not enzymes).

 

For the rest, standard procedure is creating overexpression vectors, introduce them to suitable strains, overexpress and then purify them.

[Mark Ian]

so I'm guessing you don't have any protocols...? I thought the purification process would be enzyme specific in the purification step. I am familiar with the standard protocol for overexpression & introduction steps, but I never purified enzymes before.

Edited August 28, 2012 by Mark Ian

[CharonY]

It is pretty much the same, however you have to avoid denaturing conditions. Most of the time the kits work perfectly alright (Hist-tags are pretty standard, for example).

I have dozens of protocols, but they depend quite a bit on what you want to purify and the principles are much better documented in standard protocol books (which I would recommend to beginners). Check out Molecular Clonin (Sambrook) for instance.

[alpha2cen]

Purification is a key factor to make commercial product. To make much enzyme, column chromatography method is useful. In most case, purification is more difficult than making enzyme. Paper chromatography is simple, but the enzyme obtained there is very small.

[CharonY]

Preparative LC is not terribly tricky (well establishing good condition can be a little bit). I would disagree that the upstream steps are easy in comparison. Getting the proteins to fold correctly can be a bloody nightmare. Also, in many cases additional purification via HPLC is not necessary.

Edited August 29, 2012 by CharonY