Chimaeras of the Rubisco Large Subunit from Wheat and Anacystis do not Assemble into Active Enzyme in E. coli

A new restriction site was engineered in the cloned gene codingfor the large subunit polypeptide of ribulose 1, 5-bisphosphatecarboxylase (Rubisco) of the cyanobacterium Anacystis nidulans.This change resulted in the mutation of a phenylalanine residueto an isoleucine residue in the encoded polypeptide but hadno effect on the assembly or biochemical properties of Rubiscocontaining the polypeptide. The mutation was in a loop regionlinking highly structured domains at the N and C termini ofthe complete large subunit. Using the new restriction site, and a corresponding EcoRl restrictionsite in the cloned gene for the native large subunit polypeptideof wheat Rubisco, chimaeric genes were made encoding the polypeptidewith either the 140 residues of the N-terminal part of the wheatlarge subunit fused to the 336 residues forming the C-terminalregion of the A. nidulans large subunit, or the alternativeof 136 residues comprising of the N-terminal chains of A. nidulanssubunit and the 338 residue chain at the C-terminus of the wheatlarge subunit polypeptide. The chimaeric proteins expressedin E. coli, together with the small subunit of the A. nidulansRubisco, formed an insoluble inactive aggregate mainly in inclusionbodies. The possible reasons for the failure to obtain activeholoenzyme are discussed. Key words: Rubisco, protein engineering, site-directed mutagenesis