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.
References
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
tzodikov@princeton.edu
