title: Expression of rat pro cholecystokinin (CCK) in bacteria and in insect cells infected with recombinant baculovirus.
abstract: Neuropeptide prohormones are generally not abundant in nature as they exits to be processed and contain sites which could be cleaved by a number of cellular proteases. In order to study the processing of prohormones in vitro it is necessary to produce them in quantity. Finding an expression system which produces intact prohormone has been a matter of trial and error. We report that intact rat pro CCK was produced with an amino-terminal His-Tag in e. coli, and it was secreted from sf9 and other insect cells infected with a recombinant baculovirus vector. The bacteria contained about 0.1 micrograms pro CCK/ml of cells. The High 5 insect cells produced 4.3 micrograms/ml medium (as determined by RIA), 10 times as much as sf9 or sf21. Using a combination of ion exchange, gel filtration and HPLC, the insect cell protein was purified about 150 fold with a recovery of about 16%. The secreted insect cell pro CCK is tyrosine sulfated like its mammalian equivalent. Using these expression systems it is possible to produce significant (microgram to mg) quantities of pro CCK for immunologic, enzymatic and structural studies.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Yum
authors.forename: M C
authors.initials: MC
authors.lastname: Beinfeld
mesh_terms: D000818:Animals; D001419:Bacteria / Q000821:virology*; D016367:Baculoviridae / Q000235:genetics*; D002766:Cholecystokinin / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors; D007313:Insecta / Q000821:virology*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; C060249:procholecystokinin; D002766:Cholecystokinin