Abstract
Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protetin of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 111-125 |
| Number of pages | 15 |
| Journal | Methods in Enzymology |
| Volume | 559 |
| DOIs | |
| State | Published - Jun 20 2015 |
| Externally published | Yes |
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Keywords
- CBD
- Cell harvesting
- Cell lysis
- Chitin beads
- IMPACT™-System
- Intein/Chitin binding protein tags
- Protein affinity purification
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
Protein Affinity Purification using Intein/Chitin Binding Protein Tags. / Mitchell, Sarah F.; Lorsch, Jon R.
In: Methods in Enzymology, Vol. 559, 20.06.2015, p. 111-125.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Protein Affinity Purification using Intein/Chitin Binding Protein Tags
AU - Mitchell, Sarah F.
AU - Lorsch, Jon R.
PY - 2015/6/20
Y1 - 2015/6/20
N2 - Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protetin of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.
AB - Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protetin of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.
KW - CBD
KW - Cell harvesting
KW - Cell lysis
KW - Chitin beads
KW - IMPACT™-System
KW - Intein/Chitin binding protein tags
KW - Protein affinity purification
UR - http://www.scopus.com/inward/record.url?scp=84931570256&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84931570256&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2014.11.002
DO - 10.1016/bs.mie.2014.11.002
M3 - Article
VL - 559
SP - 111
EP - 125
JO - ImmunoMethods
JF - ImmunoMethods
SN - 1046-2023
ER -