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Title: Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli

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{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.

Authors:
; ;  [1]
  1. National Research Council Canada, Montreal, Quebec (Canada)
Publication Date:
OSTI Identifier:
6217517
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology; (USA)
Additional Journal Information:
Journal Volume: 56:9; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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

Citation Formats

Romeyer, F M, Jacobs, F A, and Brousseau, R. Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli. United States: N. p., 1990. Web.
Romeyer, F M, Jacobs, F A, & Brousseau, R. Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli. United States.
Romeyer, F M, Jacobs, F A, and Brousseau, R. 1990. "Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli". United States.
@article{osti_6217517,
title = {Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli},
author = {Romeyer, F M and Jacobs, F A and Brousseau, R},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/6217517}, journal = {Applied and Environmental Microbiology; (USA)},
issn = {0099-2240},
number = ,
volume = 56:9,
place = {United States},
year = {Sat Sep 01 00:00:00 EDT 1990},
month = {Sat Sep 01 00:00:00 EDT 1990}
}