Random Mutagenesis

Philipp Wechner philipp.wechner at uibk.ac.at
Thu Jun 27 04:16:49 EST 2002



Emir Khatipov schrieb:

> Philipp,
> You say essential residues, which I understand that you have an idea of the
> protein's function. If so, I would look at the known functional motifs and
> mutate the residues in those first.

I know that there is absolutly nothing known about motives in this proteine.
All that i know is 4 essential residues.


> You can also try using those protein
> structure prediction tools from expasy and identify structurally important
> regions (strands, helices, etc.), instead of just mutating every residue.

I did some expasy stuff on my protein - it could not really help me - different
methods lead to very different secondary structure predicitions.

My protien is a protease, but no protease motives were found.... it is a very
tricky protein, so i would like to identify essential residues by random
mutagenesis with a fitting screening method - i think about the reverse two
hybrid system in yeast. If my protease is still active after random mutagenesis
the yeast could grow - all nonfunctional mutants could be filtered out like
this - i hope to end up with "some" mutants of the protein that are still
active - then sequence the DNA and see which residues are deleted/changed...
these residues are not essential....
this would give mi a first idea about wich secondary structure prediction might
be best. after that i can change specific residues by site directed
mutagenesis.


>
> You might also be able to identify the residues that form contacts with each
> other, meaning that those contacts might be important for maintaining
> structural integrity of the protein. Terminal residues would probably be
> less important that internal ones. Etc., etc. This is just a suggestion.
> Hope it is helpful, although it is not exactly the answer to your question.
> Cheers,
> Emir

thanks for your suggestions - more are welcome

>
>
> "Philipp Wechner" <philipp.wechner at uibk.ac.at> wrote in message
> news:3D173F3A.F687E222 at uibk.ac.at...
> > Hello to all of you.
> >
> > I have a protein-coding sequence of 600 bp and I would like to random
> > mutagenesis with this sequence to find essential residues of the
> > Protein.
> >
> > does anybody have experience with random mutagenesis kits? I know the
> > Diversify PCR Random Mutagenesis Kit from clontech, but I never tried
> > it. Do you know how it works and how many different mutants you can get
> > with it? Are there better methods and kits around?
> >
> > Any help is welcome.
> >
> > greetings
> >
> >




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