A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli |
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| Affiliation: | 1. Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India;2. Institute of Marine Biology, National Taiwan Ocean University, Keelung 20224, Taiwan;3. School of Life Science and Technology, Shanxi University, Taiyuan 030006, China;4. Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy;5. Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy |
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| Abstract: | ![]()
Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220–224], was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme. This construction entailed removing all lacZ-coding sequences and a portion of the 5'-noncoding leader of the R. rubrum rbc gene. The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R. rubrum ribosome binding site and the rbc structural gene. The carboxylase expressed in E. coli JM107 was purified to near homogeneity and, based on subunit Mr and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R. rubrum. N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same. |
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