Preparation and properties of recombinant rat and human procholecystokinin(57-95).
Recombinant human progastrin(6-80) binds two ferric ions with an apparent dissociation constant of 2.2 +/- 0.1 microM [Baldwin (2004) Protein J 23:65-70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin(57-95) were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin(57-95) did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin(57-95) was at least 40-fold weaker than the binding of ferric ions to progastrin(6-80).
Friedrich
Susann
S
Department of Surgery, Austin Health, University of Melbourne, Heidelberg, VIC 3084, Australia.
Sims
Yulia
Y
Baldwin
Graham S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002766:Cholecystokinin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D005290:Ferric Compounds; D011498:Protein Precursors; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins; C060249:procholecystokinin; D002766:Cholecystokinin