Chapter title |
Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP)
|
---|---|
Chapter number | 1 |
Book title |
Split Inteins
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6451-2_1 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6449-9, 978-1-4939-6451-2
|
Authors |
Dongli Guan, Zhilei Chen, Guan, Dongli, Chen, Zhilei |
Abstract |
Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn(2+)) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use. |
Mendeley readers
Geographical breakdown
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Unknown | 8 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 2 | 25% |
Student > Ph. D. Student | 1 | 13% |
Student > Doctoral Student | 1 | 13% |
Student > Master | 1 | 13% |
Unknown | 3 | 38% |
Readers by discipline | Count | As % |
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Immunology and Microbiology | 1 | 13% |
Unknown | 4 | 50% |