Details
| Originalsprache | Englisch |
|---|---|
| Seiten (von - bis) | 109-115 |
| Seitenumfang | 7 |
| Fachzeitschrift | Biotechnology and Applied Biochemistry |
| Jahrgang | 25 |
| Ausgabenummer | 2 |
| Publikationsstatus | Veröffentlicht - 1997 |
| Extern publiziert | Ja |
Abstract
The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (≤1 mg/ml) in the preparative mode, Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins, leading to a decrease of recovery and purity of the fusion protein. Reduction of host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent. This, in combination with 0.5-I M NaCl in the adsorption buffer, assured a purity >95% and a total protein recovery of ~34% in the preparative mode. Contamination of the product with about 2 mol of Ni(II)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6. Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(II) adsorber. One passage of Ni(II)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(II) content in the (His)6EcoRV fractions below the detection limit (~0.02 mg/l) of the atomic-adsorption spectrophotometer.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biotechnologie
- Chemische Verfahrenstechnik (insg.)
- Bioengineering
- Biochemie, Genetik und Molekularbiologie (insg.)
- Molekularmedizin
- Ingenieurwesen (insg.)
- Biomedizintechnik
- Immunologie und Mikrobiologie (insg.)
- Angewandte Mikrobiologie und Biotechnologie
- Pharmakologie, Toxikologie und Pharmazie (insg.)
- Wirkstoffforschung
- Chemische Verfahrenstechnik (insg.)
- Prozesschemie und -technologie
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in: Biotechnology and Applied Biochemistry, Jahrgang 25, Nr. 2, 1997, S. 109-115.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Purification of (His)6EcoRV [recombinant restriction endonuclease EcoRV fused to a (His)6 affinity domain] by metal-chelate affinity chromatography
AU - Oswald, Tanja
AU - Hornbostel, Gabi
AU - Rinas, Ursula
AU - Birger Anspach, F.
PY - 1997
Y1 - 1997
N2 - The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (≤1 mg/ml) in the preparative mode, Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins, leading to a decrease of recovery and purity of the fusion protein. Reduction of host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent. This, in combination with 0.5-I M NaCl in the adsorption buffer, assured a purity >95% and a total protein recovery of ~34% in the preparative mode. Contamination of the product with about 2 mol of Ni(II)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6. Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(II) adsorber. One passage of Ni(II)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(II) content in the (His)6EcoRV fractions below the detection limit (~0.02 mg/l) of the atomic-adsorption spectrophotometer.
AB - The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (≤1 mg/ml) in the preparative mode, Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins, leading to a decrease of recovery and purity of the fusion protein. Reduction of host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent. This, in combination with 0.5-I M NaCl in the adsorption buffer, assured a purity >95% and a total protein recovery of ~34% in the preparative mode. Contamination of the product with about 2 mol of Ni(II)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6. Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(II) adsorber. One passage of Ni(II)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(II) content in the (His)6EcoRV fractions below the detection limit (~0.02 mg/l) of the atomic-adsorption spectrophotometer.
UR - http://www.scopus.com/inward/record.url?scp=0030903249&partnerID=8YFLogxK
U2 - 10.1111/j.1470-8744.1997.tb00422.x
DO - 10.1111/j.1470-8744.1997.tb00422.x
M3 - Article
AN - SCOPUS:0030903249
VL - 25
SP - 109
EP - 115
JO - Biotechnology and Applied Biochemistry
JF - Biotechnology and Applied Biochemistry
SN - 0885-4513
IS - 2
ER -