title: Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.
abstract: Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.
authors.affiliation: Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Breer
authors.forename: Beata
authors.initials: B
authors.lastname: Wielgus-Kutrowska
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Girstun
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Staroń
authors.forename: Mariko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Sadao
authors.initials: S
authors.lastname: Hikishima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yokomatsu
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bzowska
mesh_terms: D000818:Animals; D002151:Calorimetry; D002417:Cattle; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019271:Hypoxanthine / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008024:Ligands; D017510:Protein Folding; D011683:Purine-Nucleoside Phosphorylase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013154:Spleen / Q000201:enzymology; D013816:Thermodynamics
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D019271:Hypoxanthine; D011683:Purine-Nucleoside Phosphorylase