CLONING, SEQUENCE AND OVEREXPRESSION OF THE THERMOPHILIC CYANOBACTERIUM GENE FOR THE RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE

The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were cloned from a thermophilic cyanobacterium, Synechococcus sp. strain a-1. These two genes were located tandemly on the same strand of chromosomal DNA with a 467 bp spacer region. The rbcL gene codes for 474 amino acid residues (1,422 bp), and rbcS for 118 residues (354 bp). The deduced amino acid sequence for the large subunit was highly similar to those of other cyanobacteria and higher plants. The RubisCO genes were overexpressed (> 30% of total soluble protein) in E. coli cells by using pKK223-3 as an expression vector. The overexpressed RubisCO formed hexadecamers (L8S8 form) in E. coli cells, the same form as in Synechococcus cells. The RubisCO was easily purified from E. coli cell free extract using the advantage of its high thermostability, and the purified RubisCO had almost the same characteristics as the native RubisCO purified from Synechococcus sp. strain a-1.