Cloning and Identification of a cDNA from Rat Liver Coding for a Portion of L-Gulonolactone Oxidase

Abstract

An enzyme, L-gulono-γ-lactone oxidase, was purified from the livers of male albino rats and used to prepare a polyclonal rabbit'antiserum. Reaction of the enzyme in cell-free extracts with the antiserum gave a single precipitin line when tested by the double diffusion method on agarose plates. Using the purified enzyme, the antiserum could detect nanogram quantities of the enzyme in dotblotting procedures. This antiserum was used to screen a rat liver cDNA library in the vector λgtll. Positively reacting clones could easily be identified. One clone contained a cDNA insert of 800 bp and produced a β-galactosidase fusion protein with a MW 30, 000 greater than the native molecule.