Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli
- National Research Council Canada, Montreal, Quebec (Canada)
The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB{prime}::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3 kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd{sup 2+} and Cu{sup +} (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd{sup 2+} and Cu{sup +} (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.
- OSTI ID:
- 6217517
- Journal Information:
- Applied and Environmental Microbiology; (USA), Vol. 56:9; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
METALLOTHIONEIN
DNA-CLONING
BIOLOGICAL ACCUMULATION
CADMIUM COMPOUNDS
COPPER COMPOUNDS
ESCHERICHIA COLI
GENE OPERONS
GENETIC ENGINEERING
MUTANTS
NEUROSPORA
SALMONELLA TYPHIMURIUM
BACTERIA
CLONING
DNA HYBRIDIZATION
EUMYCOTA
FUNGI
HYBRIDIZATION
METALLOPROTEINS
MICROORGANISMS
ORGANIC COMPOUNDS
PLANTS
PROTEINS
SALMONELLA
TRANSITION ELEMENT COMPOUNDS
550400* - Genetics