Larimer F W, Machanoff R, Hartman F C
Gene. 1986;41(1):113-20. doi: 10.1016/0378-1119(86)90273-8.
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.
大肠杆菌质粒pRR36可表达作为融合蛋白的深红红螺菌核酮糖二磷酸羧化酶/加氧酶（EC 4.1.1.39）[纳冈等人，《分子与普通遗传学》193（1984）220 - 224]，该质粒被用于构建羧化酶基因（rbc）的一个新克隆，其表达产物为野生型酶。此构建过程需要去除深红红螺菌rbc基因的所有lacZ编码序列以及部分5' - 非编码前导序列。用一个将trp - lac（tac）杂合启动子与深红红螺菌核糖体结合位点及rbc结构基因并列的表达载体，观察到了羧化酶的最高比活性。在大肠杆菌JM107中表达的羧化酶被纯化至接近均一，基于亚基相对分子质量和比酶活性，分离得到的蛋白质似乎与来自深红红螺菌的天然核酮糖二磷酸羧化酶没有区别。对克隆酶的N端序列分析证实克隆酶与野生型酶相同。
