Princeton University Office of Technology Licensing and Intellectual Property

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Simple, Economical Bioseparation Technique Using Self-Cleaving Thermally Responsive Fusion Tags, Princeton Invention # 04-2188

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.  This method relies on a self-cleaving elastin-like polypeptide (ELP) fusion tags, which effectively eliminate 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 ten structurally diverse proteins with high purity, activity and reasonable yield.

Development of simple and reliable methods for protein purification is an important goal for both large-scale production of purified recombinant proteins and high throughput proteomics studies of peptide libraries.  Self cleaving ELP tags consisting of repeating pentapeptides of VPGXG (X=any amino acid) fused to a controllable self-cleaving intein have the potential to be an essential tool in bioseparations.

Researchers at Princeton have used a tightly controllable ELP tag with an engineered self-cleaving intein to create a simple, economical alternative to conventional methods of protein purification. As shown in published work, the target protein gene is fused to a gene encoding a self-cleaving ELP-intein tag, and the resulting fusion is overexpressed in E. coli. The soluble ELP fusion precursor is then isolated from the insoluble cell debris by centrifugation at a temperature below the ELP T. The ELP-tagged fusion is then heated in the presence of salt to a temperature above its T, which causes the ELP tags to self associate into an insoluble precipitate. The precipitated ELP fusion can then be separated from the soluble cellular components in a second centrifugation step, and redissolved in a pH 6.0 buffer at low temperature. The intein self-cleavage reaction then releases the target protein from the ELP tag, which can be subsequently removed by an additional cycle of salt addition, heating and centrifugation. Thus a highly purified native protein is produced from a total cell lysate via a 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.

References : 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. Patent protection is pending.

For more information on Princeton University Invention # 04-2188 please contact:

                                Laurie Tzodikov

                                Office of Technology Licensing and Intellectual Property

                                Princeton University

                                4 New South Building

                                Princeton, N J 08544-0036

                                (609) 258-7256

                                (609) 258-1159 fax

                                tzodikov@princeton.edu