Modification with a phosphorylation tag of PKA in the TraT-based display vector of Escherichia coli

We have previously developed the TraT display system to express the preS1 peptide of human hepatitis B virus (HBV) and the snake venom rhodostomin (RHO) on the surface of Escherichia coli. In this study, we modified the pT2 vector by adding a thrombin cutting site and a phosphorylation tag of protein kinase A before the multiple restriction enzyme sites. The modified vector allowed us to label the TraT fusion protein (TraT-RHO) with [P-32] and to increase the detection sensitivity of TraT-RHO expression bacteria binding to and being internalized into BHK-21 cells. After the thrombin cleavage, the isotope labeled RHO could be detected in a free form. We therefore suggest that the new version of pT2 vector, pT2-KL, will facilitate to identify the counterpart of displayed peptide. (C) 2000 Elsevier Science B.V. All rights reserved.