Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli.
A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide. Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus. A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes. When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min. Its size on sucrose gradients indicates a monomeric or dimeric form. When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur. The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits. This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.
Gatenby
A A
AA
Plant Breeding Institute, Cambridge.
van der Vies
S M
SM
Rothstein
S J
SJ
D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000235:genetics*; D012995:Solubility; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D004247:DNA; D012273:Ribulose-Bisphosphate Carboxylase