Purification of E. coli Proteins Using a Self-Cleaving Chitin-Binding Affinity Tag
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The use of affinity tags to purify recombinant proteins is ubiquitous in molecular biology. However, tag removal after purification still remains a challenge. The most commonly used method, proteolytic digestion, has several drawbacks that make the process complex and costly. One alternative to the use of proteolytic digestion is the use of self-cleaving purification tags. Here, we describe a system that combines a chitin-binding domain (CBD) tag with the ∆I-CM intein to yield a self-cleaving purification tag. A protein gene of interest is genetically fused downstream of the tag, generating a fusion protein that can be rapidly and easily purified using a chitin resin. Intein self-cleavage is then induced by a simple pH and temperature shift, liberating the free target protein. This system can be used to readily purify any recombinant protein that can be expressed in E. coli, and has the potential to be applied to a wide variety of additional tags and expression hosts.
Key wordsProtein purification Intein Affinity chromatography Self-cleaving tag E. coli Purification platform Chitin binding domain
This work was supported by Army Research Office Grant W911NF-11-1-0118.
- 1.Kimple ME, Sondek J (2004) Overview of affinity tags for protein purification. Curr Protoc Protein Sci Chapter 9:Unit 9 9. doi: 10.1002/0471140864.ps0909s36
- 7.Fong BA, Wu WY, Wood DW (2010) The potential role of self-cleaving purification tags in commercial-scale processes. Trends Biotechnol 28(5):272–279, doi: 10.1016/j.tibtech.2010.02.003. S0167-7799(10)00033-8 [pii]
- 9.Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA, Pelletier JJ, Paulus H, Xu MQ (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192(2):271–281, doi:S0378-1119(97)00105-4 [pii]PubMedCrossRefGoogle Scholar
- 10.Chong S, Montello GE, Zhang A, Cantor EJ, Liao W, Xu MQ, Benner J (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res 26(22):5109–5115, doi:gkb815 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
- 17.Banki MR, Gerngross TU, Wood DW (2005) Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci 14(6):1387–1395, doi:ps.041296305 [pii]. 10.1110/ps.041296305 PubMedCentralPubMedCrossRefGoogle Scholar
- 18.Gallagher SR (2012) One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Mol Biol Chapter 10:Unit 10 12A. doi: 10.1002/0471142727.mb1002as97