Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails
(1996) In Biotechnology and Bioengineering 51(2). p.8-221- Abstract
Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine... (More)
Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity.
(Less)
- author
- Carlsson, Jonas ; Mosbach, K LU and Bülow, L LU
- organization
- publishing date
- 1996-07-20
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biotechnology and Bioengineering
- volume
- 51
- issue
- 2
- pages
- 8 pages
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:18624332
- scopus:15844412754
- ISSN
- 0006-3592
- DOI
- 10.1002/(SICI)1097-0290(19960720)51:2<221::AID-BIT12>3.0.CO;2-P
- language
- English
- LU publication?
- yes
- id
- 12284f14-388b-4e17-a360-3c1d6a782697
- date added to LUP
- 2016-04-18 16:02:02
- date last changed
- 2024-01-03 23:54:16
@article{12284f14-388b-4e17-a360-3c1d6a782697,
abstract = {{<p>Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity.</p>}},
author = {{Carlsson, Jonas and Mosbach, K and Bülow, L}},
issn = {{0006-3592}},
language = {{eng}},
month = {{07}},
number = {{2}},
pages = {{8--221}},
publisher = {{John Wiley & Sons Inc.}},
series = {{Biotechnology and Bioengineering}},
title = {{Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails}},
url = {{http://dx.doi.org/10.1002/(SICI)1097-0290(19960720)51:2<221::AID-BIT12>3.0.CO;2-P}},
doi = {{10.1002/(SICI)1097-0290(19960720)51:2<221::AID-BIT12>3.0.CO;2-P}},
volume = {{51}},
year = {{1996}},
}