Abstract
The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Antibodies / immunology
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Antibodies / isolation & purification
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Blotting, Western
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Cell Line
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Cholecystokinin / biosynthesis*
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Cholecystokinin / chemistry
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Cholecystokinin / isolation & purification
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Chromatography, Gel
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Chromatography, High Pressure Liquid
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Culture Media, Conditioned / chemistry
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Culture Media, Serum-Free
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Drosophila / cytology
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Gene Expression
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Genetic Vectors / genetics
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Histidine / genetics
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Histidine / isolation & purification
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Mass Spectrometry
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Molecular Weight
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Polymerase Chain Reaction
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Protein Precursors / biosynthesis*
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Protein Precursors / chemistry
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Protein Precursors / isolation & purification
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Rats
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Transfection
Substances
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Antibodies
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Culture Media, Conditioned
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Culture Media, Serum-Free
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Protein Precursors
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Recombinant Proteins
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polyhistidine
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Histidine
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procholecystokinin
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Cholecystokinin