Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage

Biotechnology and Bioengineering
Fang LiuWilfred Chen

Abstract

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 r...Continue Reading

References

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Citations

Nov 12, 2015·Biotechnology Journal·Zhanglin LinXu Wang
Dec 18, 2013·Biotechnology Advances·Ana Sofia PinaAna Cecília A Roque
May 27, 2014·Current Opinion in Structural Biology·David W Wood
Mar 29, 2014·Journal of Biotechnology·Mark B van EldijkJan C M van Hest
Jun 30, 2015·Chemical Communications : Chem Comm·Qi ChenWilfred Chen
Mar 14, 2018·Nature Chemistry·Rebecca P ChenWilfred Chen
Jul 10, 2019·Biotechnology and Bioengineering·Heejae KimWilfred Chen
Jun 7, 2020·Current Protein & Peptide Science·Vibhor Mishra
Oct 27, 2021·Biotechnology and Bioengineering·Yikun HuangYu Lei

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