Forschungsdatenbank der Universität Zürich Medizinische Fakultät Biochemisches Institut Prof Dr Andreas Plückthun In vitro evolution of protein function affinity stability and folding

							Forschungsdatenbank der Universität Zürich
Medizinische Fakultät > Biochemisches Institut > Prof. Dr. Andreas Plückthun



In vitro evolution of protein function: affinity, stability and folding efficiency

Summary / Zusammenfassung
One of the most powerful strategies to improve the properties of proteins or even create new ones
is to imitate the strategy of evolution in the test tube. Evolution is an iteration between
diversification and selection. The selectable function (binding) needs to be made dependent on the
property to be selected. With "ribosome display", a method has been developed over the last years
in which whole functional proteins can now be enriched in a cell-free system for their binding
function, without the use of any cells, vectors, phages or transformation (Proc. Natl. Acad. Sci. 94,
4937, 1997; J. Immunol. Meth. 231, 119, 1999; FEBS Lett., 450, 105, 1999; Adv. Protein Chem.
55, 367, 2000; Trends Biochem. Sci. 26, 577, 2001; J. Immunol. Methods 290, 51, 2004).
Correctly folded proteins can be selected, if the folding of the protein on the ribosome is secured
(Nat. Biotechnol. 15, 79, 1997). This system has been demonstrated to constitute a library
selection method for antibodies and other proteins with a "built-in" affinity maturation (Proc. Natl.
Acad. Sci. 95, 14130, 1998; Nat. Biotechnol. 18, 1287, 2000; Proc. Natl. Acad. Sci. U.S.A. 98, 75,
2001; J. Biol. Chem. 279, 18870, 2004; Nat. Biotechnol. 22, 575, 2004). This technology of
directed evolution over many generations is currently being exploited in a number of projects to
address fundamental questions of protein structure and stability (Proc. Natl. Acad. Sci. U.S.A. 98,
75, 2001; FEBS Lett. 539, 24, 2003), catalysis (Nat. Biotechnol. 21, 679, 2003) as well as
interesting biomedical applications. The affinity of antibodies was increased to KDs in the range
of 5-100 pM in several independent experiments (Proc. Natl. Acad. Sci. 95,14130, 1998; Proc.
Natl. Acad. Sci. U.S.A. 98, 75, 2001; Proc. Natl. Acad. Sci. U.S.A. 98, 8572, 2001; J. Biol. Chem.
279, 18870, 2004), as was the affinity of designed ankyrin repeat proteins (Nat. Biotechnol. 22,
575, 2004). A general protocol for establishing such high affinities was developed. Recently, an
anti-prion antibody could be evolved to an affinity of 1 pM (Luginbühl et al. 2006). Alternatively,
the stability of antibodies was improved by first destabilizing the protein by removing or reducing
the disulfide bond and then selecting for compensatory mutations (J. Mol. Biol. 283, 95, 1998,
Proc. Natl. Acad. Sci. U.S.A. 98, 75, 2001). Another approach, in conjunction with phage display,
was to select at high temperature (J. Mol. Biol. 294, 163, 1999; Cur. Opin. Struct. Biol. 9, 514,
1999). It could be demonstrated that even those proteins can be selected that cannot be expressed
at all in vivo (Protein Eng. Des. Sel. 18, 285, 2005; J. Mol. Biol. 352, 229, 2005).
Weitere Informationen unter http://www.bioc.uzh.ch/plueckthun/index.php?pid=2-0-0

Publications / Publikationen
P. Amstutz, H. Koch, H. K. Binz, S. Deuber and A. Plückthun, Rapid Selection of Specific MAP
Kinase-Binders from Designed Ankyrin Repeat Protein Libraries. Protein Eng. Des. Sel., in press
(2006).
B. Schimmele and A. Plückthun, Identification of a functional epitope of the Nogo receptor by a
combinatorial approach using ribosome display. J. Mol. Biol. 352, 229-241 (2005).
B. Schimmele, N. Gräfe and A. Plückthun, Ribosome display of mammalian receptor domains.
Protein Eng. Des. Sel. 18, 285-294 (2005).
P. Amstutz, H. K. Binz, P. Parizek, M. T. Stumpp, A. Kohl, M. G. Grütter, P. Forrer and A.
Plückthun, Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin
repeat proteins. J. Biol. Chem. 280, 24715-24722 (2005).
H. K. Binz, P. Amstutz, A. Kohl, M. T. Stumpp, C. Briand, P. Forrer, M. G. Grütter and A.
Plückthun, High-affinity binders selected from designed ankyrin repeat protein libraries, Nat.


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Biotechnol. 22: 575-582 (2004).
D. Lipovsek and A. Plückthun, In-vitro protein evolution by ribosome display and mRNA display,
J. Immunol. Methods 290: 51-67 (2004).
C. Zahnd, S. Spinelli, B. Luginbühl, P. Amstutz, C. Cambillau and A. Plückthun, Directed in vitro
evolution and crystallographic analysis of a peptide binding scFv antibody with low picomolar
affinity, J. Biol. Chem. 279: 18870-18877 (2004).
S. Cesaro-Tadic, D. Lagos, A. Honegger, J. H. Rickard, L. J. Partridge, G. M. Blackburn and A.
Plückthun, Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic
antibody library, Nat. Biotechnol. 21: 679-685 (2003).
T. Matsuura and A. Plückthun, Selection based on the folding properties of proteins with ribosome
display, FEBS Lett. 539: 24-28 (2003).
P. Amstutz, J. N. Pelletier, A. Guggisberg, L. Jermutus, S. Cesaro-Tadic, C. Zahnd and A.
Plückthun, In vitro selection for catalytic activity with ribosome display, J. Am. Chem. Soc. 124:
9396-403 (2002).
C. Schaffitzel, I. Berger, J. Postberg, J. Hanes, H. J. Lipps and A. Plückthun, In vitro generated
antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae
macronuclei, Proc. Natl. Acad. Sci. U.S.A. 98: 8572-8577 (2001).
L. Jermutus, A. Honegger, F. Schwesinger, J. Hanes and A. Plückthun, Tailoring in vitro evolution
for protein affinity or stability, Proc. Natl. Acad. Sci. U.S.A. 98: 75-80 (2001).
J. Hanes, L. Jermutus and A. Plückthun, Selecting and evolving functional proteins in vitro by
ribosome display, Methods Enzymol. 328: 404-430 (2000).
C. Schaffitzel, J. Hanes, L. Jermutus and A. Plückthun, Ribosome display: an in vitro method for
selection and evolution of antibodies from libraries, J. Immunol. Methods 231: 119-135 (1999).
J. Hanes, L. Jermutus, S. Weber-Bornhauser, H. R. Bosshard and A. Plückthun, Ribosome display
efficiently selects and evolves high-affinity antibodies in vitro from immune libraries, Proc. Natl.
Acad. Sci. U S A 95: 14130-14135 (1998).
J. Hanes and A. Plückthun, In vitro selection and evolution of functional proteins by using
ribosome display, Proc. Natl. Acad. Sci. U S A 94: 4937-4942 (1997).
Weitere Informationen unter http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-0-0

Keywords / Suchbegriffe
Ribosome display, in vitro translation, single-chain Fv (scFv) fragments, recombinant antibodies,
repeat proteins, DARPins, directed evolution

Project Leadership and Contacts / Projektleitung und Kontakte
Prof. A. Plückthun (Project Leader)                                         plueckthun@bioc.uzh.ch

Other Links to external Webpages / Andere Links zu externen Webseiten
http://www.bioc.uzh.ch/plueckthun/

Funding Source(s) / Unterstützt durch
SNF (Personen- und Projektförderung), SNF (Programm NFS/NCCR)




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Duration of Project / Projektdauer
Jan 1996 to Jan 2020




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