Functional Assay - DNMT's in vitro (Apr/10/2005 )

Hi everyone,

I've started a project in which we aim to measure the methylation efficiency of the DNA Methyltransferases in vitro. We are thinking of cloning, expressing and purifying the protein in bacterial cells, and using this in the assay.

Would this work? I mean, can you make an enzyme such as this, and get enough amount/activity from it? I've only really read articles where they've used part of the protein (either N or C term ends).
I've also read where DNMT3L enhances the activity. Would this be necessary?

Any help would be appreciated!
Dave

-Davo-

I don't see any reasions why your plan won't work. We use nuclear extract as a source of DNMTs for in vitro assay. I think recombinant enzymes should work better.

-pcrman-

Thanks pcrman, it is encouraging to hear that we're on the right track.

-Davo-

QUOTE (Davo @ Apr 13 2005, 09:24 AM)

Thanks pcrman, it is encouraging to hear that we're on the right track.

Hi
Maybe I did not read paper carfully.
But I think DNMTs are all large proteins.
Can these bacterial recombinat proteins work well?
Because I may also have to purify these proteins, can you tell me your results?
Should baculovirus purified proteins be batter?
Thanks very much

-CFLee-

Hi,

We are still planning our methods, so we haven't done any purification yet.
Regarding the size, PCR man suggests that this method should work.
I have found a paper in which human DNMT2 was overexpressed in E coli (Nucleic Acids Research, 2001, Vol 29, No. 2, 439-448), however DNMT2 is roughly 1/3 the size of DNMT3B, and DNMT1 is larger again.

The article has some info on their purification methods which may be helpful to you.

-Davo-