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TKB1 Cells Identify Receptor Tyrosine Kinase Interacting Proteins

3). The phosphoproteins found in the TKB1 cells expressing gT-F3 likely represented contaminating bacterial proteins, as these have not been repeatedly observed in other experiments; this suggests that TKB1 efficiently induces the tyrosine phosphorylation of GST fusion proteins but does not phosphorylate GST itself.

Figure 3

Fusion Proteins Used to Purify Signaling Molecules

As we previously determined that SHC proteins bind directly to tyrosine D on Neu (Y1227),4,8 we sought to determine whether these fusion proteins could be used to affinity purify cellular signaling molecules in vitro. Lysates from exponentially growing NIH-3T3 fibroblasts were incubated with gT-YC, gT-YD, gT-YE, and gT-F3 purified from BL21 or TKB1; specifically associated proteins were analyzed by immunoblotting with SHC specific antisera (Figure 3). SHC did not interact with fusion proteins produced in the BL21 strain, yet efficiently bound gT-YD when expressed in TKB1. The lack of binding can neither be attributed to the amounts nor the level of tyrosine phosphorylation of the fusion proteins (Figure 3, upper and middle panels). Thus, because this approach is specific, it permits rapid screenings for interactions with known proteins.

TKB1 Cells: A Superior Approach

GST-fusion proteins that harbor tyrosine phosphorylation sites can be used to affinity purify cellular signaling proteins in vitro when purified from TKB1 bacteria. These in vitro interactions are highly specific, as demonstrated by the binding of SHC protein to the same site as observed in vivo. We failed to detect proteins that do not interact with these phosphorylation sites (e.g., Ras-GAP) and successfully used this approach to map
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