phosphorus-radioisotopes and rhodostomin

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 [32P] 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.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Base Sequence; Binding Sites; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Genetic Vectors; Molecular Sequence Data; Peptides; Phosphorus Radioisotopes; Phosphorylation; Recombinant Fusion Proteins; Thrombin