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Construction and expression of functional multi‐domain polypeptides in Escherichia coli : expression of the Neurospora crassa metallothionein gene
Author(s) -
Mauro J. M.,
Pazirandeh M.
Publication year - 2000
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.2000.00697.x
Subject(s) - neurospora crassa , escherichia coli , biology , metallothionein , maltose binding protein , gene , plasmid , tandem repeat , periplasmic space , biochemistry , recombinant dna , fusion protein , gene expression , microbiology and biotechnology , genome , mutant
J.M. MAURO and M. PAZIRANDEH.2000. 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 109 Cd 2+. 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.