Refolding, purification, and characterization of a loop deletion mutant of human Bcl-2 from bacterial inclusion bodies.

摘要: This report describes the cloning of recombinant human Bcl-2, in which putative disordered loop region has been replaced with a flexible linker and hydrophobic C-terminus 6xHis tag (Bcl-2(6–32)-AAAA-Bcl-2(86–206)-HHHHHH, abbreviationrhBcl-2; amino acid numbering excludes initiating methionine). protein was expressed inEscherichia coliwhere it accumulated insoluble form inclusion bodies. After lysis washed bodies were solubilized anl-arginine assisted refolding route employed to obtain biologically active protein.rhBcl-2 purified further by nickel chelate chromatography give >95% purity, an overall yield 5 mg per g ofE. colicell paste. Edman sequencing showed that ∼90% therhBcl-2 retained methionine residue. Analytical size exclusion suggested refolded purifiedrhBcl-2 monomeric nondenaturing solution. Purified had affinity for Bax BH3 domain peptide comparable forin vivofolded Bcl-2 suppressed caspase activation cell-free assay apoptosis.1H NMR spectroscopy ofrhBcl-2, both free complexed peptide, provided evidence structural functional integrity protein. These findings parallel extend those Muchmoreet al.,who found deletion mutant Bcl-XLretained anti-apoptotic function.