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 cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes. The genomic fragment also contained an apparent truncated sequence corresponding to the C-terminus of the glgB gene (branching enzyme). The presence of active branching enzyme activity in crude sonicates of Rb. sphaeroides cells indicates that the genome contains a full-length glgB at another location. The structure of this operon in relation to other glg operons is further discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family. The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level 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 native strain. The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step.
Igarashi
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Department of Chemistry, California State University, Fullerton, Fullerton, California, 92834, USA.
Meyer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D006003:Glycogen; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase