Immunoaffinity purification of calpastatin and calpastatin constructs.
It has been difficult to purify calpastatin without using a step involving heating to 90-100 degrees C. Preparations of calpastatin obtained after heating often contain several polypeptides that have been ascribed to proteolytic degradation. Because calpastatin is highly susceptible to proteolytic degradation and several different calpastatin isoforms can be produced by using different start sites of transcription/translation and/or alternative splicing from the single calpastatin gene, it is not clear whether the different polypeptides observed in purified calpastatin preparations are proteolytic fragments or calpastatin isoforms. It would be useful, therefore, to have a method for purifying calpastatin that does not involve heating. At low ionic strength, calpastatin from skeletal muscle extracts binds quantitatively to an immunoaffinity column made by coupling a monoclonal antibody (MAb) to the C-terminal end of calpastatin (epitope between amino acids 707 and 786) to agarose; the bound calpastatin can be eluted at pH 2.5. The C-terminal end of the calpastatin polypeptide was used because the known isoforms of calpastatin all contain domain IV. The eluted calpastatin, which retains all its calpain inhibitory activity, consists largely of a 125 kDa polypeptide (70%), and several smaller polypeptides that are labeled with a MAb to calpastatin. Expressed calpastatin constructs representing the full-length XL-IV calpastatin and domains L-IV, II-IV, III-IV, and IV also bind to the immunoaffinity column and can be purified. The immunoaffinity column is especially useful for purifying calpastatin from small tissue samples in a single step.
Wei
Wei
W
Muscle Biology Group, University of Arizona, P.O. Box 210038, Tucson, AZ 85721-0038, USA.
Li
Hongqi
H
Cong
Jinyang
J
Thompson
Valery F
VF
Goll
Darrel E
DE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D003964:Diaphragm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010920:Placenta / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D020033:Protein Isoforms / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; C033668:calpastatin; D002918:Chymotrypsin