Refolding, purification, and characterization of a loop deletion mutant of human Bcl-2 from bacterial inclusion bodies.
This report describes the cloning of recombinant human Bcl-2, in which the putative disordered loop region has been replaced with a flexible linker and the hydrophobic C-terminus has been replaced with a 6xHis tag (Bcl-2(6-32)-AAAA-Bcl-2(86-206)-HHHHHH, abbreviation rhBcl-2; amino acid numbering excludes the initiating methionine). This protein was expressed in Escherichia coli where it accumulated in insoluble form in inclusion bodies. After lysis the washed inclusion bodies were solubilized and an l-arginine assisted protein refolding route was employed to obtain biologically active protein. rhBcl-2 was purified further by nickel chelate chromatography to give protein of >95% purity, with an overall yield of 5 mg per g of E. coli cell paste. Edman sequencing showed that approximately 90% of the rhBcl-2 retained the initiating methionine residue. Analytical size exclusion chromatography suggested that the refolded and purified rhBcl-2 was monomeric in nondenaturing solution. Purified protein had an affinity for a Bax BH3 domain peptide comparable to that for in vivo folded recombinant human Bcl-2 and suppressed caspase activation in a cell-free assay for apoptosis. 1H NMR spectroscopy of rhBcl-2, both free and complexed with the Bax BH3 domain peptide, provided further evidence for the structural and functional integrity of the refolded protein. These findings parallel and extend those of Muchmore et al., who found that a loop deletion mutant of human Bcl-XL retained anti-apoptotic function.
Anderson
M
M
Cancer & Infection Research Departments, Protein Structure Laboratory, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom. Malcolm.Anderson@alderley.zeneca.com
Blowers
D
D
Hewitt
N
N
Hedge
P
P
Breeze
A
A
Hampton
I
I
Taylor
I
I
D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3; D061945:Caspase Inhibitors; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D019254:Genes, bcl-2; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017384:Sequence Deletion; D014982:Xenopus laevis; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D061945:Caspase Inhibitors; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; D053148:Caspase 3