|Title:||Intracellular Delivery of Recombinant alpha B-crystallin into Neonatal Rat Cardiomyocytes has a Protective Effect on the Cells|
|Abstract:||In order to deliver alpha B-crystallin (alpha B-C) into cardiomyocytes and study its cellular protection, the full-length cDNA fragment encoding human alpha B-C was cloned into the bacterial expression vector pGEX-MTS containing the base sequence of membrane-translocating sequence (MTS) which mediates intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S-transferase (GST).After glutathione affinity chromatography and cleaved from GST by factor Xa, the recombinant MTS- alpha B-C was separated from GST and factor Xa by anion exchange chromatography. Recombinant MTS- alpha B-C was characterized by SDS-PAGE and Western immunoblot analysis. The purified MTS- alpha B-C migrated on SDS-PAGE as a single band to an apparent molecular weight (Mr.23kD) that corresponded to total native alpha B-C plus MTS, and was recognized on Western immunoblot by anti-human alpha B-crystallin antibody. MTS- alpha B-C displayed chaperone-like function in an ATP-containing buffer at 37? by disaggregating the denatured and aggregated actin induced by hydrogen peroxide (H2O2 )treatment. It was observed under fluorescence microscope that FITC-labeled MTS- alpha B-C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with it for 6 hours while FITC-labeled alpha B-C and bovine serum albumin had not gone into the cells. Recombinant MTS- alpha B-C is not cytotoxic, and MTS- alpha B-C-treated cells displayed increased H2O2-tolerance compared with non-treated cells.|
|Subject Headings:||alpha B-crystallin|
|Appears in Collections:||Original Articles|
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