cloning strategy for protein interaction using pETDuet-1 and PullDown

Dear all,

I´m testing interaction of three proteins with a bait. Our plan is to
coexpress each one of the three target proteins separately with the
bait using pETDuet-1 (contains 2 multiple cloning sites (MCS), each
cloning site contains a tag (His tag and S tag) for protein isolation
and purification).

I´m trying to devise a best strategy for coexpression followed by pull
down assays and would appreciate if someone could suggest a good
strategy for this kind of experiment.

My question is: What is the best strategy to use?

example:

1) Clone target protein A fused to a His tag into MCS1 and bait
protein fused to S tag into MCS2? Induce expression of both proteins
and separately purify protein A-his tag with a nickel resin and bait
with a S-affinity resin for pull down?


2) Clone target protein A fused to a His tag into MCS1 and bait
protein alone (no tag here) into MCS2 ? Induce expression of both
proteins and bind protein A into nickel resin. But then how do I test
interaction of protein A with bait if the bait was not purified
previously with a different tag?
Can I use lysate of co-expressed proteins to bind bait to the protein
A previously bound to nickel resin?

Any help appreciated,


Jose

Graduate student
University of CAMPINAS, UNICAMP
Bazil