Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli
- 1 September 1990
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 56 (9) , 2748-2754
- https://doi.org/10.1128/aem.56.9.2748-2754.1990
Abstract
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'::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 Cd2+ and Cu+ (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 Cd2+ and Cu+ (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.This publication has 28 references indexed in Scilit:
- Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coliGene, 1989
- Proton NMR studies of a metallothionein from Neurospora crassa: sequence-specific assignments by NOE measurements in the rotating frameBiochemistry, 1989
- Biochemistry of metallothioneinBiochemistry, 1988
- METALLOTHIONEINAnnual Review of Biochemistry, 1986
- Metallothioneins: Proteins in search of functionCell, 1985
- High-level expression of M13 gene II protein from an inducible polycistronic messenger RNAGene, 1985
- Cadmium-Resistant Pseudomonas putida Synthesizes Novel Cadmium ProteinsScience, 1984
- Metal substitution of Neurospora copper metallothioneinBiochemistry, 1984
- Prokaryotic metallothionein: Preliminary characterization of a blue-green alga heavy metal-binding proteinBiochemical and Biophysical Research Communications, 1979
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970