Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1. expression and characterization of recombinant ADP-glucose pyrophosphorylase

A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was clo ned from a genomic library using a polymerase chain reaction probe coding f or part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open, reading frames for the glg C and glgA (glycogen. synthase) genes and partial sequences corresponding t o glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) gen es. The genomic fragment also contained an apparent truncated sequence corr esponding to the C-terminus of the glgB gene (branching enzyme). The presen ce of active branching enzyme activity in crude sonicates of Rb. sphaeroide s cells indicates that the genome contains a full-length glgB at another lo cation. The structure of this operon in relation to other glg operons is fu rther discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences a nd discussed in relation to the allosteric regulation of this enzyme family . The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-le vel expression in E. coli. The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the n ative strain. The recombinant enzyme was purified to near homogeneity and f ound to be physically, immunologically, and kinetically identical to the na tive enzyme, verifying the fidelity of the cloning step. (C) 2000 Academic Press.