Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP).
Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals. The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 micromol min(-1)mg(-1)) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.
Breer
Katarzyna
K
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki & Wigury 93, 02-089 Warsaw, Poland.
Girstun
Agnieszka
A
Wielgus-Kutrowska
Beata
B
Staroń
Krzysztof
K
Bzowska
Agnieszka
A
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003460:Crystallization; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D019271:Hypoxanthine / Q000737:chemistry; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
D018076:DNA, Complementary; D010710:Phosphates; D011994:Recombinant Proteins; D019271:Hypoxanthine; D007288:Inosine; D006147:Guanine; C008450:7-methylguanine; D011683:Purine-Nucleoside Phosphorylase