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Strategy for highly...
Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner
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- Gräslund, Torbjörn (författare)
- KTH,Biokemi och biokemisk teknologi
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- Ehn, Maria (författare)
- KTH,Biokemi och biokemisk teknologi
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- Gunnel, Lundin (författare)
- KTH,Biokemi och biokemisk teknologi
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- Hedhammar, My (författare)
- KTH,Biokemi och biokemisk teknologi
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- Uhlén, Mathias (författare)
- KTH,Biokemi och biokemisk teknologi
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- Nygren, Per-Åke (författare)
- KTH,Biokemi och biokemisk teknologi
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- Hober, Sophia (författare)
- KTH,Proteomik
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(creator_code:org_t)
- 2002
- 2002
- Engelska.
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 942:1-2, s. 157-166
- Relaterad länk:
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http://www.sciencedi...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- Biochemistry
- Biokemi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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