Protein interaction cloning
Cheung C. Yue
ccy at po.CWRU.Edu
Wed Oct 7 16:34:46 EST 1992
Quite some time back, I had responded to a post by stating a number
of new techniques have been described to clone genes for proteins
which interact with other proteins. A subsequet post requested references.
My desk has been a mess and it's taken me a while to find them, so here
they are (at least a partial list).
Interaction cloning: identification of a helix-loop-helix zipper protein
that interacts with c-fos. Science 256:1014; '92.
A fusion protein is constructed with the protein of interest linked to 3
modules: a) a FLAG sequence for easy purification, b) an enterokinase
peptidase cleavage site, and c) a HMK (heart muscle kinase) recognition
site for 32-P labeling. The fusion protein is then used for screening an ex
The two-hybrid system: a method to identify and clone genes for proteins
that interact with a protein of interest. PNAS 88:9578; '91.
DNA binding domain of GAL-4 is fused to protein of interest. A library
of sequences is then fused to the activation domain of GAL-4. Functional
reconstitution of GAL-4 is read out by a reporter gene with GAL-4 binding sites.
A contingent replication assay for the detection of protein-protein
interactions inanimal cells. PNAS 88:1086; 91.
Similar to the two-hybrid system. Has an amplification loop using
SV40 T antigen cloned behind GAL-4 binding site, and the fusion protein
cloned behind SV40 ori. Newly amplified plasmid sequences are DpnI-
resistant due to lack of methylation, and are selected for by DpnI digestion.
Direct selectionfor sequences encoding protease of known specificity. PNAS
Still using GAL-4, but now introduces a specific protease target site
between the DNA binding domain and the activation domain. If specific
protease is co-expressed in the same cell, GAL-4 function would be lost,
which can then be selected for.
ccy at po.cwru.edu
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