HISTIDINE PATCH THIOREDOXINS - MUTANT FORMS OF THIOREDOXIN WITH METALCHELATING AFFINITY THAT PROVIDE FOR CONVENIENT PURIFICATIONS OF THIOREDOXIN FUSION PROTEINS

A cluster of surface amino acid residues on Escherichia coli thioredox in were systematically mutated in order to provide the molecule with a n ability to chelate metal ions, The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nicke l ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Se pharose resins, Even though these two histidines were more than 30 res idues apart in thioredoxin's primary sequence, they were found to sati sfy the geometric constraints for metal ion coordination as a result o f the thioredoxin tertiary fold, A third histidine mutation, S1H, prov ided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding, All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for conve nient purifications of human interleukin-8 and -11 expressed in E. col i as soluble thioredoxin fusion proteins.