Abstract
Recombinant human progastrin6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [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 procholecystokinin57–95 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 procholecystokinin57–95 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 procholecystokinin57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin6–80.
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Abbreviations
- CCK:
-
Cholecystokinin
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- Gamide:
-
Gastrin17
- Ggly:
-
Glycine-extended gastrin17
- PBS:
-
Phosphate-buffered saline
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Acknowledgements
We gratefully thank Margery Beinfeld (Tufts University School of Medicine, Boston, MA) and Lars Johnson (Rigshospitalet, Copenhagen, Denmark) for generous gifts of plasmids encoding rat (pCMV5) and human (pGEM11) proCCK, respectively, and Arthur Shulkes (University Department of Surgery, Austin Health) for many helpful discussions. This work was supported in part by the Austin Hospital Medical Research Foundation, and by a grant 400062 (to GSB) from the National Health and Medical Research Council of Australia.
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Friedrich, S., Sims, Y. & Baldwin, G.S. Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95 . Protein J 27, 186–191 (2008). https://doi.org/10.1007/s10930-007-9122-z
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DOI: https://doi.org/10.1007/s10930-007-9122-z