Probing of an expression library with oligopeptides.

Sai siyer at
Thu Feb 6 12:49:15 EST 1997

In article <DLC22-0602971049220001 at>, DLC22 at CORNELL.EDU
(CLARKEY) wrote:

> We are presently looking into the possibility of probing a lambda zap
> expression library with an oligo-peptide known to bind to specific
> receptor, namely Beta1 Integrin.  The oligo is RGD and is linked to
> biocytin via an SPC linker.  We will detect the probe using avidin
> conjugated to alkaline phosphotase.
> Therefore my questions are.
> 1. Is this feasible?
> 2. Has anyone tried to do this previously?
> 3. Will the expressed protein be presented to the peptide in the correct 
>    conformation?
> 4. Are there any alternatives to this procedure?
> answer to question 1, it is very feasible.  this type of screening
is known as interaction cloning (or expression cloning (?)).  what you are
doing is screening for proteins that are encoded by your cDNA expression
library that interact to your oligo-peptide probe via the interacting
protein's functional domain.  the key words here are funcitonal domain cuz
the cDNA encoding for whatever interacts with your probe may not be the
full-length cDNA of that protein/gene.  this of course, depends on the
library you are using and the average size of the insert packed in the
phage.  to continue on to question 2, this has been tried b4 many times
and currently being done by myself (with fairly good results..).  some of
the bcl2 family genes (bad or bax, i'm not sure which ones, but you can
find the refs. if you search under the keyword interaction cloning) were
cloned this way in conjunction with other methods (2 hybrid, affinity
columns etc...).  there are a bunch of variants to this approach.  the
first being that screening can be done with other labelled proteins (as
opposed to peptides), or antibodies to specific proteins (the more
conventional approach known as expression cloning).  another variant is
the far western concept (or protein overlay) where the primary probe is
the protein that you are screening with and the seocndary probe is the
antibody to the protein probe.  therefore, detection would be via the
antibody...the first variant that i mentioned is a bit mroe direct in the
sense that you can directly detect your probe that's bound thru its
radioactive label.  ok, moving onto your third question,  this depends on
if whatever protein(s) you are screening for go thru post-xlational
modifications (glycosylation, phosphorylation etc) to give them their
final functional domains that would interact with your protein probe.  if
you dont have this information, it is then an empirical process to go
ahead with the cloning procedure, obtain the clone, express it in e.coli
and mammalian systems and then carry out binding assays and structural
studies (analytical ultracentrifugation or circ.dichroism to give
secondary structural information).  you can even compare mobilities in gel
filtration assays to see if the same protein expressed in different
systems has the same approximate shape and size.  if it does have post
xlational modofications that alter its conformation or adds mass
(phosophorylation etc), that would become evident if it gets retarded
during gel filtration.  native gels would also giove you similar info
since mobility thru the acrylamide pore would be size, shape and charge
dependant since the protein stays native.  
   finally, procedures like bio-panning done with peptide probes is an
alternative to this procedure.  you can get much more info that i can give
you on bio-panning by checking the refs (search the keyword of
bio-panning).  novagen just came out with some kit that has all the
necessary stuff to let you do bio-panning.  you might wanna check that
out...good luck...cheers....

Sai Iyer
siyer at
graduate student; dept of biochemistry and molecular genetics
university of alabama at birmingham

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