A novel recombinant chemokine protein TAT-SDF-1/54-KDEL for blocking CXCR4: bioinformatics analysis and prokaryotic expression (CCR5P.263)

Abstract

Abstract To explore the potential application of SDF-1/54 mutant in Immunology, a prokaryotic vector pTAT-HA was exerted to express a novel recombinant protein ,TAT/SDF-1/54/KDEL, which contains three distinct functional domains: murine SDF-1/54, a 11-peptide TAT (47-57) derived from human immunodeficiency virus tat trans-activator protein and an endoplasmic reticulum retention four-peptide sequence KDEL.Bioinformatics analysis showed that the estimated half-life of the protein in mammalian reticulocytes,yeast,Escherichia coli in vitro are 2.8h, 10h and 2min. Respectively.Endoplasmic reticulum distribution and localization ratio is 13.0%. The data also shows the novel protein on the secondary structure and topology proteins , protein-binding sites, the three-dimensional map and a possible prediction catalytic pocket and the binding sites. Our expression experiment showed that the expected band of the protein appeared in the precipitate by SDS-PAGE electrophoresis. Western blot confirmed the specificity of the immune response of this protein to the rabbit anti-His-probe antibody.These suggest that the bioinformatics analysis for TAT/54R/KDEL might be an effective approach to predict its structure and functional expression. The expression of the protein in pTAT-HA prokaryotic vector is in the form of inclusion bodies. It offered a new method targeting for CXCR4 implicated immunity regulation,such as HIV, WHIM syndrome, cancer and stem cells mobilization and so on.