Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells.
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.
Kleditzsch
Petra
P
Institute of Pharmacology and Toxicology, Medical Faculty of the Humboldt-University Charité, 10117, Berlin, Germany. Petra.Kleditzsch@charite.de
Pratt
John
J
Vishnuvardhan
Daesety
D
Henklein
Peter
P
Schade
Rüdiger
R
Beinfeld
Margery C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002460:Cell Line; D002766:Cholecystokinin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned / Q000737:chemistry; D016895:Culture Media, Serum-Free; D004330:Drosophila / Q000166:cytology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D000906:Antibodies; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011498:Protein Precursors; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; C060249:procholecystokinin; D002766:Cholecystokinin