Simple, Economical Bioseparation Technique Using Self-Cleaving Thermally Responsive Fusion Tags

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Princeton Invention # 05-2188


Researchers in the Chemical Engineering Department at Princeton University have developed a new economical method for the purification of recombinant proteins expressed in E. coli and other expression host cells.  This method relies on a self-cleaving elastin-like polypeptide (ELP) fusion tag, which effectively eliminates the need for conventional affinity chromatography or proteolytic tag removal in the purification of arbitrary fusion proteins. Furthermore, this method allows the recovery of native product proteins, without additional amino acids on either terminus, and has been used to purify over a dozen structurally diverse proteins with high purity, activity and reasonable yield.  The generality of this method for applications with a wide variety of proteins and expression systems makes it an attractive platform technology for the study and manufacture of recombinant proteins in virtually any context.


This method is based on a tightly controllable elastin-like peptide (ELP) purification tag, combined with an engineered self-cleaving intein.  The ELP tag has the unique ability to reversibly precipitate in response to mild changes in temperature and salt concentration.  The selective precipitation of this tag is used as the basis for the purification method.  At the same time, the self-cleaving ability of the intein allows simple removal of the ELP tag once the purification procedure is complete.  As demonstrated in several published papers from this group, a target protein gene can be fused to that of the self-cleaving ELP-intein tag.  This is accomplished via simple recombinant DNA methods, and the resulting gene fusion is overexpressed in an appropriate expression host, such as E. coli or Pichia pastoris. The ELP fusion precursor is then isolated from whole cells (for secreted products) or insoluble cell debris (for cytoplasmic expression products) by simple centrifugation under conditions where the ELP tag is soluble.  Once the solution is clarified, the ELP-tagged fusion is selectively precipitated by mild heating and/or the addition of one of several salts.  The precipitated ELP and fused target protein can then be separated from the soluble impurities via a second centrifugation step.  Once purified, the ELP is resuspended in a pH 6.5 buffer to trigger the intein self-cleavage reaction, which releases the target protein from the ELP tag.  The cleaved ELP-intein tag can then be trivially removed by a final cycle of selective precipitation and centrifugation. Thus a highly purified native protein can be produced via simple mechanical means, without chromatography. 


The simple mechanical recovery of precipitated ELP fusion protein suggests a variety of means for scale-up, including tangential flow microfiltration or continuous centrifugation. Alternatively the method might be used in a robotic system to purify protein libraries for screening.  The simplicity and self-contained nature of this system promise a breakthrough in the production of purified recombinant proteins in research and industrial enzymes for commercial use.




Fong, B. A., Wu, W.-Y. & Wood, D. W., 2009, Optimization of ELP-intein mediated protein purification by salt substitution, Protein Expression and Purification, Vol. 66 (2), 198-202.


Wu, W.-Y., Fong, B. A., Gillies, A. R. & Wood, D. W., 2009, Recombinant Protein Purification by Self-cleaving Elastin-like Polypeptide Fusion Tag, Current Protocols in Protein Science, (In Press).


Gillies, A. R., Hsii, J. F., Oak, S. & Wood, D. W., 2008, Rapid cloning and purification of proteins: Gateway vectors for protein purification by self-cleaving tags (Editors┬┐ Choice feature publication), Biotechnology and Bioengineering, Vol. 101 (2), 229-240.


Mee, C., Banki, M. R. and Wood, D. W., 2008, Towards the Elimination of Chromatography in Protein Purification: Expressing Proteins Engineered to Purify Themselves, Chemical Engineering Journal, Vol. 135, 56-62.


Wu, W.-Y, Mee, C., Califano, F., Banki, R. & Wood, D.W., 2006, Recombinant Protein Purification by Self-Cleaving Aggregation Tag, Nature Protocols, Vol. 1, 2257-2262.


Banki, M.R. & Wood, D.W., 2005, Inteins and Affinity Resin Substitutes for Protein Purification and Scale Up, Microbial Cell Factories, Vol. 4:32.


Banki,M., Feng,L., Wood, D., September 2005, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nature Methods, Vol. 2 No.9, 659-661.


Princeton is currently seeking industrial collaboration to commercialize this technology.


For more information on Princeton University Invention # 05-2188 please contact:


                                Laurie Tzodikov

                                Office of Technology Licensing and Intellectual Property

                                Princeton University

                                4 New South Building

                                Princeton, NJ 08544-0036

                                (609) 258-7256

                                (609) 258-1159 fax



Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
David Wood
Mahmoud Banki