Production and purification of a recombinant human hsp60 epitope using the cellulose-binding domain in Escherichia coli.
The heat shock protein hsp60 plays a functional role in insulin-dependent diabetes mellitus. The hsp60 epitope p277 (aa 437-aa 460) is effective in vaccinating mice against diabetes. A synthetic peptide gene (p277) that encodes the human hsp60 epitope was cloned to the 3' end of the cellulose-binding domain gene (cbd). CBD-p277 was overexpressed in Escherichia coli and purified on a cellulose column. A methionine at the C-terminal end of CBD enabled CNBr cleavage between CBD and p277. After CNBr cleavage, free CBD and residual uncleaved CBD-p277 were recovered by cellulose chromatography. The p277 peptide was further purified on a RPC-FPLC column. The molecular weight of the recombinant peptide was confirmed by electrospray mass spectrometry. The recombinant peptide was found to be biologically active in assays involving clone C9 T-cell proliferation, lymph-node cell proliferation, and antibody production. Thus the use of CBD as an affinity tag and the utilization of affordable cellulose matrices offers an attractive method for the production and purification of recombinant peptides.
Shpigel
E
E
The Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, 76100, Israel.
Elias
D
D
Cohen
I R
IR
Shoseyov
O
O
D002455:Cell Division / Q000187:drug effects; D002482:Cellulose / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry*; D003488:Cyanogen Bromide / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000503:physiopathology; D000939:Epitopes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry*; D013601:T-Lymphocytes / Q000378:metabolism
D018834:Chaperonin 60; D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002482:Cellulose; D003488:Cyanogen Bromide