NAD biosynthesis systems
The present invention generally relates to a nicotinamide adenine dinucleotide (NAD) biosynthesis system and methods of screening for NAD biosynthesis effectors. Among the various aspects of the present invention is the provision of an in vitro-reconstituted mammalian NAD biosynthesis system that can be used for the high-throughput screening of chemical activators and inhibitors for mammalian NAD biosynthesis. Another aspect of the invention provides a method of identifying a compound that effects in vivo activity of NAD metabolic enzymes. Further aspects of the invention include nucleic acid sequences, vectors, and transformed cells that can be used in the methods described herein.
1. An aqueous solution for the in-vitro biosynthesis of reduced nicotinamide adenine dinucleotide (NADH) comprising:
a nicotinamide phosphoribosyltransferase (Nampt) polypeptide at a concentration of about 0.01 μg/mL to about 100 μg/mL;
a mammalian nicotinamide mononucleotide adenylyltransferase (Nmnat) polypeptide at a concentration of about 0.01 μg/mL to about 200 μg/mL;
alcohol dehydrogenase protein (ADH);
bovine serum albumen (BSA);
ATP;
phosphoribosyl pyrophosphate (PRPP);
a magnesium ion;
ethanol; and
a buffer selected from the group consisting of HEPES and Tris, wherein the Nampt and the Nmnat are each at least partially purified from other cellular constituents.