This approach is superior to other methods: A number of known candidate molecules can be quickly and simultaneously screened by immunoblot analyses and be readily modified to identify novel proteins through the use of labeled cellular extracts. Fusion proteins that contain a single tyrosine residue can be used to map sites of interaction; and interaction sites can be identified which, when mutated in vivo, lead to a loss of protein activity or stability. For example, when two tyrosine residues mutate within the juxtamembrane region of the platelet-derived growth factor receptor, the kinase is rendered catalytically inactive;9 hence, interacting proteins cannot be identified.
Use the tyrosine-phosphorylated fusions for a variety of applications: Carry out direct interactions with soluble tyrosine-phosphorylated proteins and immobilized interaction SH2/PTB domains, both produced in E. coli; screen bacteriophage expression libraries (for a nonisotopic variation of the CORT assay,10 in which novel SH2-containing proteins are cloned using a carboxy-terminal region of receptor tyrosine kinases); and generate and purify tyrosine-phosphospecific antibodies.
Alternatively, phosphotyrosine-containing peptides that are chemically synthesized can also be used to yield similar results. However, synthetic peptide reagents are expensive, nonrenewable, and require prior immobilization for such an assay (e.g., biotinylation or chemical crosslinking to a support matrix). On the other hand, tyrosine-phosphorylated fusion proteins produced in bacteria are a renewable and inexpensive resource.
Tyrosine phosphorylated and unphosphorylated polypeptides are efficiently
produced in TKB1 and BL21 bacteria, respectively. These proteins can be used to
rapidly define a