Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene.

Abstract

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and targeted to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind 109Cd2+. The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.