Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene.
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.
Mauro
J M
JM
Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC 20375, USA.
Pazirandeh
M
M
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D009842:Oligopeptides / Q000096:biosynthesis / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020080:Tandem Repeat Sequences / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D002104:Cadmium; D008668:Metallothionein