Overproduction of Anabaena 7120 ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli.
As a prerequisite to protein engineering, we have overexpressed the rbcLS operon of the cyanobacterium Anabaena 7120, in Escherichia coli. The operon encodes the large and small subunits of ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Levels of active enzyme exceed 6% of soluble protein. We noted an apparent third gene, an unidentified open reading frame (URF) referred to here as rbcX, in the 558-bp intergenic space between the large and small subunit encoding genes. The URF, rbcX, has no known function. High-level production of Rubisco activity from the rbc operon in E. coli required simultaneous overproduction of the GroESL chaperonins under a regimen of limited growth, in contrast to more modest conditions which suffice for efficient production of the Anacystis nidulans cyanobacterial Rubisco. Deletion of rbcX or inversion of the rbcL-rbcS order did not enhance expression levels. The recombinant Rubisco, purified to near homogeneity, exhibited functional properties [Km(ribulose-P2), kcat, transition-state-analogue binding stoichiometry/exchange, and specificity factor] essentially identical to those of the enzyme obtained from Anabaena.
Larimer
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Protein Engineering Program, Oak Ridge National Laboratory, TN 37831-8077.
Soper
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TS
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011994:Recombinant Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase