generating terminal truncations of a protein
dk at no.email.thankstospam.net
Thu Sep 19 21:59:03 EST 2002
"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote:
>It depends on the size of truncations and on how many do you want to
>generate at once. For short truncations QuickChange (to which I am in no way
>related) usually results in very decent rate of success.
I've had no problems deleting >250 bases with QuikChange. A paper
somewhere in BioTechniques claims there is virtually no limit to this
as they succeded to remove >2 kb. Chopping off a lot has an advantage
that in addition to DnpI one can treat reaction with a cutter a site
for which is supposed to be absent in the desired mutant. In this
case, background becomes literally zero.
>> What are the routine methods by which you generate terminal truncations
>> of a protein of interest for subsequent screening for purification,
>> stability and efficient folding for the purposes of crystallization?
>> My protein has 2 known domains and a conserved C-terminal part which is
>> defined as domain by sequence homology with other proteins. We would
>> like to generate different lengths of the protein containing this
>> C-terminus and screen them for expression, solubility, purification, and
>> folding and thus increase our chances for success.
I've heard that a nice approach to this is to make a C-terminal
GFP fusion and random prime from N-terminus. Brighly fluorescent
clones are those that have mutants in frame _and_ soluble (insoluble
product drags GFP into inclusion bodies where it self-quenches.
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