Engineering sensor proteins for antibody detection by design and evolution

171 Pages·2017·18.61 MB·English

Engineering sensor proteins for antibody detection by design
and evolution
Citation for published version (APA):
van Rosmalen, M. (2017). Engineering sensor proteins for antibody detection by design and evolution. [Phd
Thesis 1 (Research TU/e / Graduation TU/e), Biomedical Engineering]. Technische Universiteit Eindhoven.
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Published: 20/11/2017
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Engineering sensor proteins for antibody
detection by design and evolution

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Technische Universiteit
Eindhoven, op gezag van de rector magnificus prof.dr.ir. F.P.T. Baaijens,
voor een commissie aangewezen door het College voor Promoties, in het
openbaar te verdedigen op maandag 20 november 2017 om 16:00 uur

door

Martijn van Rosmalen

geboren te Leeuwarden
Dit proefschrift is goedgekeurd door de promotoren en de samenstelling
van de promotiecommissie is als volgt:

voorzitter: prof.dr. P.A.J. Hilbers
1e promotor: prof.dr. M. Merkx
2e promotor: prof.dr.ir. L. Brunsveld
leden: prof.dr. G.J. Poelarends (Rijksuniversiteit Groningen)
dr. S.G.D. Rüdiger (Universiteit Utrecht)
prof.dr.ir. M.W.J. Prins
dr. T.F.A. de Greef
adviseur: dr. J. Tel

Het onderzoek dat in dit proefschrift wordt beschreven is uitgevoerd in
overeenstemming met de TU/e Gedragscode Wetenschapsbeoefening.

Table of contents
Chapter 1 Introduction: Engineering sensor proteins 3
using directed evolution

Chapter 2 Yeast display for engineering FRET sensor 37
proteins by directed evolution

Chapter 3 Affinity maturation of a cyclic peptide 63
handle for therapeutic antibodies using
deep mutational scanning

Chapter 4 Dual color bioluminescent sensor proteins 97
for therapeutic drug monitoring of
anti-tumor antibodies

Chapter 5 Tuning the flexibility of glycine-serine 127
linkers to allow rational design of
multidomain proteins

Chapter 6 Epilogue 147

Summary 156

List of publications 160

Curriculum vitae 161

Dankwoord 162

1
2
1
C
HAPTER

Introduction: Engineering
sensor proteins using directed

evolution

Van Rosmalen, M. and Merkx, M. Engineering sensor proteins using directed
evolution, (in preparation)
3
CHAPTER 1
Abstract
Engineering fluorescent / luminescent sensor proteins by rational design is a laborious process.
Directed evolution offers a compelling alternative to rational design, but currently this strategy is
not widely adopted in the field of protein sensors. Here we review the progress that has been
made in this regard in recent years and the challenges that must still be addressed. In addition to
the lack of high-throughput screening methods for sensor proteins a major impediment to their
engineering is that each sensor requires unique target binding domains and therefore relies on a
unique sensing mechanism. More generic sensor designs would be advantageous. We review
potentially generic sensor formats that have been put forward by various groups and discuss
their pros and cons. Finally, we propose a generic platform, FRET-bodies which makes use of
insertion of hypervariable loops onto the surface of fluorescent proteins.

4
Engineering sensor proteins using directed evolution
Introduction
This thesis concerns the combination of two widely used protein engineering strategies, rational
design and directed evolution for the engineering of sensor proteins. Engineering such sensors
purely using rational approaches entails much trial and error and is a slow and tedious process.
Directed evolution, generating and screening diverse mutant libraries may be a valuable
complementary strategy. The literature review presented in this introduction serves as a
reference that places the other chapters into the proper context. Specifically, it addresses the
question which directed evolution strategies have been used so far for sensor proteins. In
addition, the concept of a generic sensor scaffold is discussed along with several examples of
such scaffolds. Chapter 2 presents an attempt to use yeast display for engineering the FRET-
sensor protein for antibody detection, AbSense, towards recognition of new target antibodies.
This proved to be unsuccessful, because the yeast displayed sensor protein adopted a low-FRET
conformation, markedly different to that of the same sensor purified from bacteria. Chapter 3
then describes the affinity maturation of a so-called ‘meditope’ peptide that specifically binds to
the therapeutic antibody cetuximab using yeast display and ‘deep mutational scanning’.
Subsequently, in chapter 4 the peptides from chapter 3 were incorporated into luminescent
sensor proteins for cetuximab detection. Several sensor proteins for detection of different
therapeutic antibodies are presented in chapter 4 as well. This two-step approach was aided by a
rational thermodynamic understanding of the sensor mechanism. Chapter 5 details the tuning of
the length and stiffness of glycine-serine linkers often used in sensors and other multidomain
proteins. Finally, the epilogue in chapter 6 provides suggestions for further research on how the
antibody sensor proteins presented in this study may be further improved using a two-step
approach of both rational protein design and directed evolution.
Genetically encoded luminescent or fluorescent sensor proteins report on the concentrations of
target molecules and thus provide a valuable window into the inner workings of the cell. (1)
Sensor proteins consist of engineered versions of naturally occurring luciferases and / or
Fluorescent Proteins (FPs) as well as ligand binding domains. Upon engaging their intended
target molecules, these sensor proteins change their luminescent or fluorescent properties.
It is difficult to exhaustively categorize sensors, as many different designs have been reported
and summarizing all of them is beyond the scope of this thesis. However, in general terms
sensors can be grouped in several different ways by their sensing mechanism. A first distinction
can be made between fluorescent and luminescent sensor proteins. Fluorescent sensor proteins
require and external excitation light source for fluorescence, while luminescent sensor which
employ luciferases require the presence of a substrate in order to function. This distinction has
important practical consequences. Because fluorescence is much brighter than luminescence,
these sensors are in principle better suited for microscopy applications. Fluorescent sensors are
also the preferred choice if fast kinetics (<1 s) are required, such as for monitoring membrane
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