generating terminal truncations of a protein
AEVDOKIMOZ at cinci.rr.com
Thu Sep 19 19:56:29 EST 2002
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. You might also
achieve reasonable success with site mutagenesis of the start and stop
codons - it is easier for the C-terminus because stop only requires one
mutation, whereas start needs two (old start out, new start in). There are
also randomized PCR-based methods for generation of random truncations on
both ends, these require the user to separate the resulting clones and
analyze them by sequencing in order to establish their identity. If you have
no ideas as to how much to cut and where from, randomization of termini
followed by mini-expressions to verify solubility and activity might be the
way to go.
"Iva Toudjarska" <toudjarska at wi.mit.edu> wrote in message
news:3D8A09FE.CE9027C4 at wi.mit.edu...
> Hi guys,
> 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.
> appreciate all help
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