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 [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.
Chang
H
H
Institute of Microbiology, School of Life Science, National Yang-Ming University, Taipei, Taiwan ROC.
Lo
S J
SJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins; D013917:Thrombin / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; C036297:TraT protein, E coli; C072026:rhodostomin; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin