Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.
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.
Breer
Katarzyna
K
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
Wielgus-Kutrowska
Beata
B
Girstun
Agnieszka
A
Staroń
Krzysztof
K
Hashimoto
Mariko
M
Hikishima
Sadao
S
Yokomatsu
Tsutomu
T
Bzowska
Agnieszka
A
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
D008024:Ligands; D011994:Recombinant Proteins; D019271:Hypoxanthine; D011683:Purine-Nucleoside Phosphorylase