Cloning, Expression, Purification, and Some Properties of Calf Purine Nucleoside Phosphorylase

Abstract

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.

Keywords:

• Calf spleen PNP
• PCR amplification
• Mammalian wild type PNP

Acknowledgments

Supported by Polish Ministry of Science and Higher Education N301 003 31/0042.