Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage
Article first published online: 17 MAY 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 11, pages 2829–2835, November 2012
How to Cite
Liu, F., Tsai, S.-L., Madan, B. and Chen, W. (2012), Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage. Biotechnol. Bioeng., 109: 2829–2835. doi: 10.1002/bit.24545
- Issue published online: 24 SEP 2012
- Article first published online: 17 MAY 2012
- Accepted manuscript online: 7 MAY 2012 07:22AM EST
- Manuscript Accepted: 23 APR 2012
- Manuscript Revised: 2 APR 2012
- Manuscript Received: 19 DEC 2011
- NSF. Grant Numbers: CBET1116090, CBET0965953
While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin–dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829–2835. © 2012 Wiley Periodicals, Inc.