An in vivo approach to isolating allosteric pathways using hybrid multimeric proteins.

Hybrid tetramers of Escherichia coli phosphofructokinase (EC 2.7.1.11; EcPFK) have been used to dissect the complicated allosteric interactions within the native tetramer. The method used previously to generate hybrids in vitro involves dissociation of the parent proteins with KSCN followed by re-association as KSCN is removed via dialysis. However, this procedure is time consuming and is plagued with low hybrid yields. Consequently, we have attempted to produce hybrids more quickly and with potentially higher yields in vivo by co-expressing the parental EcPFK protein in E. coli. Wild-type EcPFK gene was cloned into pALTER-Ex2 and pALTER-1, respectively. Site-directed mutagenesis was performed to make mutant EcPFK gene in pALTER-1. Since each vector has a different origin of replication and antibiotic selection marker, we were able to co-transform both plasmids to competent E. coli cells. Following an affinity purification column, anion-exchange chromatography was used to separate the five hybrid species (4:0, 3:1, 2:2, 1:3, 0:4). While all five hybrid species were obtained, the amount 1:3 and 0:4 hybrids were very small. By changing the expression vector for the mutant EcPFK protein from pALTER-1 to pALTER-Ex1 and the charge-tag mutations from K2E/K3E to K90E/K91E, the yield of 1:3 hybrid was substantially increased. The in vivo method does increase the yield of the hybrids produced while decreasing the time required for their isolation.