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

Lett Appl Microbiol. 2000 Feb;30(2):161-6. doi: 10.1046/j.1472-765x.2000.00697.x.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters*
  • Amino Acid Sequence
  • Base Sequence
  • Cadmium / metabolism
  • Carrier Proteins / metabolism
  • Escherichia coli / chemistry
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Maltose-Binding Proteins
  • Metallothionein / biosynthesis
  • Metallothionein / chemistry
  • Metallothionein / genetics*
  • Metallothionein / metabolism
  • Molecular Sequence Data
  • Monosaccharide Transport Proteins*
  • Neurospora crassa / genetics*
  • Neurospora crassa / metabolism
  • Oligopeptides / biosynthesis
  • Oligopeptides / metabolism
  • Plasmids / genetics
  • Protein Engineering*
  • Recombinant Fusion Proteins / biosynthesis
  • Tandem Repeat Sequences / genetics*

Substances

  • ATP-Binding Cassette Transporters
  • Carrier Proteins
  • Escherichia coli Proteins
  • Maltose-Binding Proteins
  • Monosaccharide Transport Proteins
  • Oligopeptides
  • Recombinant Fusion Proteins
  • maltose transport system, E coli
  • Cadmium
  • Metallothionein