Aus dem Department für Biomedizinische Wissenschaften
der Veterinärmedizinischen Universität Wien
Institut für Tierzucht und Genetik, Abteilung Molekulare Genetik
Leitung: O.Univ.-Prof. Dr.med.vet. Mathias Müller
Biotinylation of tyrosine kinase 2
in cell culture
to identify interaction partners
Diplomarbeit
Veterinärmedizinische Universität Wien
vorgelegt von
Kerstin Wunderl
Wien, Mai 2015
6
Betreuer: O.Univ.-Prof. Dr.med.vet. Mathias Müller
Institut für Tierzucht und Genetik, Abteilung Molekulare Genetik
Department für Biomedizinische Wissenschaften
Veterinärmedizinische Universität Wien
Gutachter: Univ.-Prof. Dr.med.univ. Veronika Sexl
Institut für Pharmakologie und Toxikologie
Department für Biomedizinische Wissenschaften
Veterinärmedizinische Universität Wien
Supervision: Dr.med.vet. Ursula Reichart
Institut für Tierzucht und Genetik, Biomodels Austria
Department für Biomedizinische Wissenschaften
Veterinärmedizinische Universität Wien
7
Table of contents: 1. Introduction 1 1.1 The role of TYK2 in the JAK/STAT signalling pathway 1 1.1.2 The structure of TYK2 1 1.2 The JAK/STAT signalling pathway 2 1.3 TYK2 as a component of cytokine receptor complexes 3 1.4 Cytokines which induce TYK2 mediated signalling 4 1.4.1 IFNs type I and III (IFN α/β and IFN λ) 4 1.4.2 IL-10 and IL-22 5 1.4.3 IL-12 and IL-23 6 1.4.4 IL-6 family 6 1.5 Affinity tags 6 1.5.1 Main characteristics of affinity tags 6 1.5.2 Advantages and drawbacks of frequently used affinity tag systems 8 1.5.2.1 Polyhistidine-tags (HIS-tag) 8 1.5.2.2 FLAG-tag 8 1.5.2.3 Strep-tag II 8 1.5.2.4 Calmodulin-binding peptide (CBP) 9 1.5.2.5 Gluthathione S-transferase (GST) 9 1.5.2.6 Maltose-binding protein (MBP) 9 1.5.2.7 Elastin-like polypeptides (ELP) 10 1.5.2.8 SUMO, NusA, Trx and FATT 10 1.6 The AviTag/BirA system 13 1.6.1 Biotinylation 13 1.6.2 Biotin protein ligase (BPL) 14 1.6.3 AviTag/BirA tagging 15 1.7 Purification of biotinylated proteins 15 1.8 Aim of the study 16 2. Material and Methods 17 2.1 Cells 17 2.1.1 TYK2 deficient murine embryonic fibroblasts (MEFs) 17 2.1.2 2fTGH wild-type cells 17
8
2.1.3 U1A cells 17 2.2 Standard cell culture procedures 17 2.2.1 Splitting of cells 18 2.2.2 Freezing of cells 18 2.2.3 Thawing of cells 18 2.2.4 Counting of cells 19 2.3 Transient transfection of cells 19 2.4 Selection of stably transfected MEFs 19 2.5 Plasmids 20 2.6 Protein analysis 20 2.6.1 Lysing of cells 20 2.6.2 Determination of protein concentration 21 2.6.3 SDS-polyacrylamide gel electrophoresis (PAGE) 21 2.6.4 Western Blot analysis 21 2.6.4.1 Stripping of blotted membranes 23 2.7 Purification of biotinylated TYK2 with Neutravidin 23 2.7.1 Separation of biotinylated protein 23 2.7.2 Elution of biotinylated protein 23 2.7.3 Precipitation of proteins 24 2.8 Proof of functionality of tagged TYK2 by Interferon stimulation 24 2.9 List of materials and reagents 25 3. Results 26 3.1 Generation of MEFs stably co-expressing Tyk2/AviTag and BirA 26 3.1.1 Serial transfection of the AviTag/BirA constructs into MEFs 26 3.1.2 Co-transfection of Tyk2/AviTag and BirA/FLAG into MEFs 27 3.1.3 Validation of the stable genome integration of Tyk2/AviTag and
BirA/FLAG 28 3.2 Purification of neutravidin-bound biotinylated TYK2 protein 29 3.3 Validation of the Tyk2/AviTag protein 32 4 Discussion 34 5 Summary 36 5.1 Zusammenfassung 37 6 References 38 7 Appendix 49 7.1 Vector map of pTyk2/AviTag-puro 49
9
7.2 Vector map of pBirA/FLAG-blast 50
List of frequently used abbreviations:
aa amino acid
BCCP biotin carboxyl carrier protein
BirA biotin protein ligase of E.coli
BPL biotin protein ligase
CoA Coenzym A
E.coli Escherichia coli
IFN interferon
TYK2 tyrosine kinase 2
IL interleukin
JAK janus kinases
MEFs murine embryonic fibroblast cells
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
SOCS suppressor of cytokine signalling
STAT signal transducer and activator of transcription
TNF tumor necrosis facto
1
1. Introduction
1.1 The role of TYK2 in the JAK/STAT signalling pathway
The tyrosine kinase (TYK) 2 is a member of the Janus-kinase/signal transducer and activator
of transcription (JAK/STAT) pathway, which is required to transmit information from
extracellular cues through transmembrane receptors directly to the nucleus, and provides a
mechanism to modify transcriptional expression without second messengers (Aaronson and
Horvath 2002).
The JAK/STAT pathway is involved in various cellular functions like proliferation,
differentiation, migration and apoptosis. Hence, it plays a crucial role in cancer, infection,
inflammation, allergy and autoimmune diseases, which makes it an ideal target for therapeutic
applications (Strobl et al. 2011, O´Shea et al. 2015).
1.1.2 The structure of TYK2
TYK2 is a member of the Janus kinase (JAK) family, which consists of four kinases: JAK1,
JAK2, JAK3 and TYK2. JAK1 and JAK2 are ubiquitously expressed, whereas JAK3 is
predominantly expressed in hematopoietic cells. They all range from 120-140kDa. JAKs are
receptor associated tyrosine kinases and important mediators of cytokine signalling (Pellegrini
and Dusanter-Fourt 1997).
The murine tyk2 gene is located on chromosome 9 and encodes a 4.4kb transcript. The TYK2
protein comprises about 1200 amino acids and displays a structure which is common to all
JAKs. They are multi-domain proteins, divided into four structural domains (kinase,
pseudokinase, SH2 and FERM) and seven homology regions (JH1-7) (Rane et al. 2002).
The N-terminus of the protein contains an SRC-Homology-2 domain (SH2) (JH3 and part of
JH4) and the so called FERM domain, which extends from JH7 to half of JH4. The SH2-
domain mediates protein-protein interactions by binding phosphorylated tyrosine residues
(Strobl et al. 2011) (Fig.1). The FERM domain is a four-point-one, ezrin, radixin, moesin
(FERM) homology domain, which appears to be critical for interaction of Janus kinases with
their cognate receptors and regulatory proteins. It is also required for the interaction of TYK2
with the interferon (IFN) α/β receptor chain IFNAR1 (Yeh and Pellegrini 1999).
2
Furthermore, JAKs contain tandem kinase domains at the carboxyl terminus. One of them is
quite similar to kinase domains, but does not show any catalytic activity (Yeh and Pellegrini
1999) and is therefore referred to as pseudokinase domain (JH2). JH1 is an active kinase
which is necessary for the phosphor-transfer activity of Janus kinases (Yeh and Pellegrini
1999).
Figure 1. Schematic depiction of the structural (FERM, SH2, pseudokinase, kinase) and functional (JH1 – JH7)
domains of TYK2. (Strobl et al. 2011)
1.2. The JAK/STAT signalling pathway
The JAK/STAT pathway provides a membrane to nucleus mechanism for the prompt
alteration of gene expression. When ligands bind to the extracellular domain of the receptor
complex JAKs get activated. Two different kinds of receptor complexes exist: For ligands
such as growth hormones or erythropoietin, the subunits of the receptor are bound as
homodimers, while for interferons or interleukins, the receptor subunits are heteromultimers
(Rawlings et al. 2004). JAKs then phosphorylate the receptor chains, which become docking
sites for STATs. The recruited STATs get activated by phosphorylation and translocate to the
nucleus where they activate or repress the transcription of target genes (Fig.2) (Levy and
Darnell 2002).
3
1.3 TYK2 as a component of cytokine receptor complexes
TYK2 plays a crucial role in cytokine signalling and host immunity. Besides its contribution
to IFN α/β signalling, it has also been discovered to be involved in IL-12 and IL-23 signalling
as well as in other cytokine signalling pathways (Fig. 3).
Figure 2. Cytokine-binding to the receptor causes tyrosine phosphorylation. JAKs get activated, followed
by the recruitment of STATs. STATs get phosphorylated and enter the nucleus as dimers to drive
transcription (Levy and Darnell 2002).
4
Figure 3. Cytokine receptors which use TYK2 for signalling (Strobl et al, 2011).
The way in which TYK2 alters cytokine signalling has been discovered by using a mutant
sarcoma cell line (U1A) lacking TYK2 (Pellegrini et al. 1989, Velazquez et al. 1992).
TYK2 deficient mice have been generated by different approaches such as conventional
knock out technology (Karaghiosoff et al. 2000, Shimonda 2000, Sheehan et al. 2006),
conditional knock-out models via Cre/loxP recombination (Vielnascher et al. 2014) and the
inactivation of the TYK2 kinase domain (Prchal-Murphy et al. 2012).
A naturally occurring mouse strain (B10.Q/J) with a mutation in the TYK2 pseudokinase
domain serves as a further possibility to explore TYK2 functions. Whether TYK2 is absent or
inactive in the mice stays a controversial issue (Shaw et al. 2003, Bieber et al. 2010).
1.4 Cytokines which induce TYK2 mediated signalling
1.4.1 IFNs Type I and III (IFN α/β and IFN λ)
The type I interferon (IFN)-family consists of about 20 members which share a structural
homology of some degree (Decker et al. 2005). The most prominent members of the family
5
are IFN α and IFN β, which also are the main interferons that are synthesized during a viral or
bacterial infection (Bogdan et al. 2004).
IFNs can be distinguished according to their cell-surface receptors: Type I IFNs α and β use
the type I IFN receptor (IFNAR) which consists of two subunits, IFNAR1 and IFNAR2.
Recently, TYK2 was also shown to be involved in IFN III signalling. IFN III lambda binds to
the subunits IFN LR1 and IL10 R2. TYK2 is associated to IFNAR1 as well as to IL10 R2
(Dornhoff et al. 2011, Kotenko 2011).
In the human sarcoma cell line U1A, which is deficient in TYK2 signalling, a complete
unresponsiveness to IFN α and a reduced but not completely abolished responsiveness to IFN
β could be observed (Velazquez et al. 1992).
This disability to respond to type I IFNs may be a result of a rigorous reduction of IFNAR1
expression and the following loss of high affinity IFN α binding in the absence of TYK2
(Ragimbeau 2003).
U1A cells also served to discover that catalytically active TYK2 is required for IFN β induced
STAT3 and IFNAR1 phosphorylation. In contrast, the enzyme activity of TYK2 is not needed
for STAT1 and STAT2 phosphorylation (Rani et al. 1999).
1.4.2 IL-10 and IL-22
IL-10 is very important for the down regulation of inflammatory responses. It is produced by
various immune cells like macrophages, dendritic cells, monocytes, B cells and T cells. It is
able to inhibit the production of pro-inflammatory cytokines, like IL-1, IL-12 or TNF-α (Riley
et al. 1999).
IL-10 receptors are tetramers consisting of two IL-10R1 polypeptide chains and two IL-10R2
chains, which associate with JAK1 and TYK2 respectively. Upon binding of IL-10 STAT1,
STAT3 and STAT5 get activated (Riley 1999). In monocytes, IL-10 is able to induce de novo
synthesis of suppressor of cytokine signalling (SOCS) 3, which inhibits gene-expression in
the cell by interfering with JAK/STAT dependant signalling (Donnelly et al. 1999).
IL-22 is an important cytokine of activated T cell subsets and innate lymphoid cells. It
transduces its effects via a heterodimeric receptor consisting of IL-22R1 and IL-10R2. With
respect to the JAK-STAT pathway JAK1, TYK2 and STAT3 are activated. IL-22 mainly
6
targets epithelial cells and hepatocytes, where it promotes proliferation, tissue regeneration
and barrier functions. However, it plays also a role in (auto-)inflammatory conditions
(Dudakov et al. 2015, Wolk et al. 2011).
1.4.3 IL-12 and IL-23
IL-12 and IL-23 are heterodimeric cytokines that consist of a p40 subunit combined with a
p35 or a p19 subunit, respectively. Both cytokine receptors use the TYK2 associated IL-
12Rbeta1 chain. The JAK2 bound subunit is unique to the respective receptor, IL-12Rbeta2
for the IL-12 receptor and IL-23-R for the IL-23 receptor (Watford et al. 2004).
STAT1, STAT3, STAT4 and STAT5 are activated by IL-12 and IL-23. In the absence of
TYK2, T-cells, NK-cells and dendritic cells cease to produce IFN γ in response to IL-12 and
therefore, STAT3 and STAT4 activation is compromised (Karaghiosoff et al. 2000).
IL-23 induces IFN γ production in DC-cells and IL-17 expression in gamma-delta T cells and
Th17-cells. These processes are also strongly impaired by TYK2 deficiency (Tokumasa et al.
2007, Nakamura et al. 2008).
1.4.4 IL-6 family
The family of IL-6 cytokines consists of cytokines signalling through the receptor subunit
gp130. IL-6, IL-11, leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary
neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) are known members of this family.
Although the requirement of TYK2 for signal transduction of these cytokines is not clear for
all species and cell types, they all induce TYK2 phosphorylation (Strobl et al. 2011 and refs
therein).
1.5 Affinity tags
1.5.1 Main characteristics of affinity tags
By definition, affinity tags are exogenous amino acid sequences with a high affinity for a
certain chemical or biological ligand (Arnau et al. 2005).
7
Affinity tags allow different proteins to be purified using one common method and
uncharacterized proteins to be efficiently and specifically purified without any prior
knowledge of their chemical properties. As a consequence they have become a widely used
and important tool in several areas of research (Arnau et al. 2005).
As there are numerous different tags, each of them has its pros and cons.
In general affinity tags can have positive effects by improving protein yields (Rajan et al.
1998), preventing proteolysis (Wang et al. 1997), facilitating protein refolding (Shi et al.
2005), protecting the antigenicity of the fusion protein (Mayer et al. 2004), or increasing
solubility (Chen et al. 2005).
But affinity tags can also negatively affect the protein of interest. The protein conformation
can be changed (Chant et al. 2005), the enzymatic activity can be inhibited (Kim et al. 2001),
protein yields can decrease (Goel et al. 2000), toxicity can occur (de Vries et al. 2003) and the
biological activity of the protein can be changed (Arnau et al. 2005).
An important factor whether the tag has an effect on the tertiary structure or on the biological
activity of the target protein is the tags amino acid composition and its location within the
protein (Bucher et al. 2002).
An ideal affinity tag would have the following properties: it would allow to efficiently purify
the target protein in high yields, could be applied to any protein of interest, would have no or
a minimal effect on the protein function, could be placed at any location within the protein, is
usable in any host strain or expression system, allows detection of the target protein and could
be bound and eluted from an inexpensive carrier substance (Lichty et al. 2005).
Short fusion tags like Polihistidine (His)-tags, Strep-tags or Calmodulin-binding peptide
(CBP) are normally used for purification of the protein of interest or to permit detection of
fusion partners. Larger tags such as Glutathione S-transferase (GST) or Maltose-binding
protein (MBP) are commonly used to enhance proper folding or solubility or to immobilise
the target protein (Kay et al. 2009).
However, best results can only be obtained if a specific purification method for a specific
protein is highly advanced in its level of optimization (Arnau et al. 2005). As there is not one
8
ideal affinity tag, the idea of combinatorial tagging has emerged. To gain the maximum
possible benefit from affinity tags, dual- and even multi-tagging systems have been developed
(Waugh 2005).
1.5.2 Advantages and drawbacks of frequently used affinity tag systems
1.5.2.1 Polihistidine-tags (His-tags)
As His-tags are quite short (5-15 aa), the activity of the target proteins is rarely affected
(Porath 1992). Other advantages of His-tags are their low metabolic burden, the inexpensive
affinity resin and the mild elution conditions. The tag works under native as well under
denaturing conditions (Waugh 2005).
The most import weakness is that proteins with external His-residues tend to co-purify with
the His-tagged target protein and therefore cause contamination. His-tags can also affect the
protein-folding and have been reported to sometimes have an effect on protein function
(Wood 2014).
1.5.2.2 FLAG-tag
The FLAG-tag, a hydrophilic 8-amino acid peptide, is used for antibody-based purification of
proteins. It has proven to be useful in various kinds of cells (bacterial, yeast, mammalian)
(Terpe 2002).
Advantages of the FLAG-tag are its relatively low metabolic burden and its high specificity.
The high costs of the affinity resin and the harsh elution conditions can be seen as
disadvantages (Waugh 2005).
1.5.2.3 Strep-tag II
Strep-tag II is an octapeptide that recognizes streptavidin with a high binding specificity.
Tagged proteins are purified using the affinity of streptavidin and are eluted with biotin
(Arnau et al. 2005, Terpe 2002)
However, it is not clear yet if the Strep-tag II interferes with protein crystallization (Lichty et
al. 2005)
9
1.5.2.4 Calmodulin-binding peptide (CBP)
CBP consists of 26 amino acid residues which derive from the C-terminus of the skeletal
muscle myosin light-chain kinase. This kinase binds calmodulin in the presence of calcium
chloride with high affinity (Blumenthal et al. 1985). Because of the tight binding, more
stringent washing conditions can be applied and thus co-purification of contaminating
proteins occurs rarely.
The CBP can be fused either at the N- or C-terminus of the protein. Placing the CBP at the C-
terminus can result in high expression levels, whereas locating it at the N-terminus may
reduce the efficiency of translation (Zheng 1997).
CBP is not recommended in eukaryotic cells, as there are endogenous proteins which interact
with calmodulin (Head 1992).
1.5.2.5 Glutathione S-transferase (GST)
GST is a protein that consists of 211 amino acid residues. As GST-tags are rather large in size
(26kDa), they tend to have a relevant impact on their fusion partners (Wood 2014).
GST-tags increase the solubility of their fusion partners and their very high specificity (Wood
2014). They are useful in the protection against intracellular protease cleavage and thus act as
a stabilizer for the recombinant proteins (Terpe 2002).
As GST is a homodimer it can complicate the purification of proteins. That renders GST
unsuitable for the isolation of oligomeric proteins (Waugh 2005).
1.5.2.6 Maltose-binding protein (MBP)
MPB, a protein of 40kDa, is composed of 370-396aa residues. MBP is a native E.coli protein
and plays a crucial part in the catabolism of maltodextrins.
Fusion of MBP to a target protein allows the one-step purification of the protein of interest by
using amylose resin (Terpe 2002).
Insoluble proteins can be recovered in a soluble and properly folded form by using the MBP
tag (Kapust and Waugh 1999).
10
1.5.2.7 Elastin-like polypeptides (ELP)
ELPs are composed of a Val-Pro-Glyx-Xaa-Gly (VPGXG) pentapeptide repeated up to 120
times. Xaa is a “guest residue” and can be any amino acid except Pro (Bidwell III and
Raucher 2005).
They can be used for temperature-induced aggregation, to separate the protein of interest from
others by centrifugation and resolubilization. As ELP-tags are usually large in size, the actual
length of the tag influences the protein yield (Meyer et al. 2001).
1.5.2.8 SUMO, NusA, Trx and FATT
When heterologous proteins are generated in Eschericha coli, they often aggregate as
insoluble folding intermediates, also called inclusion bodies. One option to avoid this is to use
GST or MBP (see above). Alternatively the solubility with tags such as NusA (N-utilization
substance A), SUMO (small ubiquitin-related modifier), Trx (E.coli thioredoxin) or FATT
(Flag acid target tag) (Terpe 2002) can be increased.
All these tags do not directly take part in any purification methods and therefore must be used
in combination with other affinity tags, but provide additional functions:
SUMO, a 100aa residue protein, is used to enhance overall expression and folding of the
target protein together with improving protein solubility. The use of SUMO is only
recommended in E.coli, as there are SUMO-proteases present in eukaryotic cells that could
interfere with protein fusion (Arnau et al. 2005).
NusA is quite large as it consists of 495aa. In comparison to MBP, NusA achieves the same
or even better results in large scale screening tests, although the performance highly varies
from protein to protein (Busso et al. 2005). NusA is often used in combination with His-tags.
Trx is a thermostable, 12kDa intracellular protein. Trx is used to avoid inclusion bodies in
recombinant proteins. For further purification steps, His-tags are commonly fused to the Trx-
tag (Terpe 2002).
11
Including a FLAG-tag module for easy detection alongside a hyperacidic segment from the
human amyloid precursor protein extracellular region, the FATT presents a notable and highly
efficient new tagging system. FATT has shown to increase the proper folding of target
proteins significantly and can be easily purified with a single step and a conventional
chromatography resin (Wood 2014).
12
Table 1 summarizes the characteristics of the different tag systems described above.
Table 1: Advantages and limitations of some commonly used tags tag type example size Advantages Limitations
His His 5-15 (usually 6)
low metabolic burden
small and inexpensive contaminants possible
minimal effect on target protein does not enhance solubility
functions under native and denaturing conditions can interfere with protein folding and function
well established
Peptide/ Epitope tags
FLAG 8 low metabolic burden Expensive
high specificity harsh elution conditions
Strep II 8
high specificity
can interfere with protein crystallization minimal target impact
exceptional purity
CBP 26 high specificity not to use in eukaryotic cells
high expression levels
Peptide on Plasmid Avi 15
minimal impact on target protein Expensive
high efficiency and specificity must be co-expressed with BirA
Folded domain tags
GST 201 - 211 mild elution condition inexpensive
high metabolic burden
slow binding kinetics
homodimeric protein
can decrease protein yields
MBP 396
enhances solubility large in size
Inexpensive high metabolic burden
mild elution conditions can interfere with target function
Precipitation/ Aggregation tags ELP 18-320
Inexpensive
large in size can influence protein yield
high expression yields
reversible transition from soluble/insoluble through temperature
Solubility/ Folding tags
NusA 495 enhance solubility can aid in refolding
must be used in combination with other tags relatively high metabolic burden SUMO 100
Trx 109
13
1.6 The AviTag/BirA system
The AviTag/BirA system utilizes a naturally occurring reaction, the biotinylation, for tagging
proteins of interest. Unlike most other tagging systems it was established as a tool for tagging
proteins ex vivo in mammalian cells and in in vivo mouse models, respectively (Driegen et al.
2005, de Boer et al. 2003).
1.6.1 Biotinylation
Because of the high efficiency and specificity of the biotinylation reaction, alongside its
virtual irreversibility, numerous scientific applications like gene-probes, immune- assays,
protein blotting or protein purification methods have emerged (Waldrop 2015). Biotin, or
Vitamin H, is a small molecule that has the extraordinary ability to bind avidin/streptavidin
with an association constant of 1015Lmol-1. That is the tightest non-covalent interaction
known in nature.
Biotin serves as a co-factor for enzymes and
participates in a number of different metabolic
pathways (Waldrop 2015). Mammals need to
obtain biotin through dietary sources or from
their intestinal bacteria, which – besides plants
and some fungi – are able to synthesize biotin de
novo (Pendini et al. 2008).
Biotin is composed of two five-membered
carbon rings and a valeric acid side chain. It´s
chemical structure is C10H16N2O3S1 and it has
a molecular weight of 224,31g/mol. Eight
possible stereoisomers are known (Waldrop 2015) (Fig. 4).
Biotin acts as a covalent carrier of carbon dioxide, therefore biotin-dependant enzymes are
referred to as carboxylases, decarboxylases and transcarboxylases.
In mammals only five enzymes get biotinylated: acetyl CoA carboxylase 1 and 2, pyruvate
carboxylase, propionyl CoA carboxylase and 3-methylcrotonyl CoA carboxylase. They are
Figure 4: Chemical structure of biotin (http://www.chemspider.com)
14
involved in fatty acid synthesis and oxidation, gluconeogenesis and the amino acid catabolism
(Waldrop 2015).
In general, biotin is primarily found covalently attached to these enzymes inside the cell. It is
a two-step post-translational reaction of very high specificity. Only bound biotin is
biologically active. The ligase that works as a catalyser for these reactions is called biotin
holoenzyme synthetase or biotin protein ligase (BPL) (Chapman-Smith and Cronan 1999,
Lane et al. 1964).
1.6.2 Biotin protein ligase (BPL)
The biotin protein ligase (BPL), also called holocarboxylase synthethase, catalyses the
attachment of biotin to the biotin-dependent enzymes. The interaction between BPL and
biotin is highly conserved in nature. BPLs can be interchanged between organisms without
losing their function, but show the highest affinity for their original substrates (Pendini et al.
2008).
The most prominent BPL is BirA, the BPL of E. coli. It is a monomeric protein of 35.3kDa
(Chapman-Smith and Cronan 1999).
BirA catalyses the biotin attachment to the biotin carboxyl carrier protein (BCCP) subunit of
the acetyl Coenzym A (CoA) carboxylase complex. Acetyl CoA carboxylase is the only
biotin-dependent enzyme found in E.coli (Waldrop 2015). BirA also represses the function of
the biotin biosynthetic operon by synthesizing its own co-repressor biotinoyl-5´adenosine
monophosphate (AMP) (Chapman-Smith and Cronan 1999).
By truncation studies Beckett et al. (1999) revealed a 14aa peptide deriving from the original
BCCP as the minimal substrate for efficient BirA activity.
Utilizing a “Direct Evolution” approach, i.e. screening peptide libraries, a consensus peptide
for the enzymatic reaction of BirA was identified (Schatz 1993), which was optimized to the
15aa AviTag by the company Avidity (www.avidity.com).
15
1.6.3 AviTag/BirA tagging
The AviTag is a short (15aa) peptide tag with the sequence GLNDIFEAQKIEWHE.
The central lysine residue gets specifically and efficiently biotinylated by the bacterial BirA
biotin ligase (Driegen et al. 2005, Kay et al. 2009).
Recombinant proteins fused to the AviTag are efficiently biotinylated in vitro by BirA. The
actual benefit of this tagging system is based on its application in in vivo situations. If the
AviTag and BirA are co-expressed in vivo, a specific and efficient biotinylation occurs in
bacteria, yeast, insect and mammalian cells (Kay et al. 2009 and refs therein)
Biotinylated proteins deriving from the AviTag/BirA system can be purified using the high
affinity of biotin for avidin and streptavidin (Rybak et al. 2004).
1.7 Purification of biotinylated proteins
Avidin, a glycoprotein found in egg yolk and streptavidin a protein produced by the bacterium
Streptomyces avidinii, can bind up to four biotin molecules.
The binding of both avidin and streptavidin with biotin is one of the tightest non-covalent
associations known in nature (Fig. 5) (Waldrop 2015).
Figure 5: Biotin – Avidin binding (http://www.lifetechnologies.com)
16
The high affinity allows purification under high stringency conditions and as there are very
few biotinylated proteins occurring naturally in the cell, the chance for cross-reaction is very
low (de Boer et al. 2003).
Deglycosylated avidin (neutravidin) showed to combine beneficial characteristics of the
natural molecules with a minimal risk for unspecific binding (Hiller et al. 1990). Table 2
compares the most common biotin binding proteins.
Table 2: Comparison of biotin-binding proteins (http://www.lifetechnologies.com)
Avidin Streptavidin Neutravidin Protein
Molecular Weight 67kDa 53kDa 60kDa
Biotin-binding Sites 4 4 4
Isoelectric Point (pI) 10 6.8-7.5 6.3
Specificity Low High Highest
Affinity for Biotin 10-15M 10-15M 10-15M
Nonspecific Binding High Low Lowest
1.8 Aim of the study
As a member of the Janus kinase family and a key player in cytokine signalling, TYK2
receives more and more attention as a target for the therapeutic intervention in the treatment
of numerous diseases. Due to the lack of a commercially available antibody for murine
TYK2, its detection in cells and tissue stays a challenge.
The aim of this study was to prove the suitability of the AviTag/BirA system for the detection
of TYK2 in in vivo systems by introducing it into TYK2 deficient primary cells to facilitate
further approaches to discover interaction partners of TYK2.
17
2. Materials and Methods
2.1. Cells
Cells were cultured using standard medium (see 2.2), unless otherwise indicated. Reagents
not listed in the cell culture reagents list (2.9) were ordered from Sigma (St.Pölten, Austria) or
Merck (Darmstadt, Germany).
2.1.1. TYK2 deficient murine embryonic fibroblasts (MEFs)
TYK2 deficient murine embryonic fibroblasts (MEFs) were obtained from stocks provided by
the institute. Shortly, fibroblasts were obtained from murine embryos at day 13.5 of
pregnancy (Karaghiosoff et al. 2000). TYK2 deficient embryonic fibroblast (MEF) cell lines
were generated by spontaneous immortalization in longterm culture (unpublished).
2.1.2. 2fTGH wild-type cells
2fTGH cells originate from the human sarcoma cell line HT1080. A genetic modification
allows the regulation of 2fTGH cells by interferons (Pellegrini et al. 1984)
2.1.3. U1A cells
The chemical mutagenesis of 2fTGH cells yielded in several mutants showing different
defects in the interferon signalling. The mutant U1A cell line is deficient in signalling by
tyrosine kinase (TYK) 2 (Pellegrini et al 1984, Velazquez et al. 2001).
2.2. Standard cell culture procedures
Cells were cultured in tissue culture plates (Corning) and incubated in humidified atmosphere
at 37°C and 5% CO2 using the standard culture medium.
The standard culture medium consisted of the following ingredients:
DMEM (high glucose 4.5g/l) (GE Healthcare) supplemented with 10% fetal calf serum
(FCS) (Life Technologies), 50μM β-mercaptoethanol (Life Technologies), 2mM L-Glutamine
(GE Healthcare), 100μg/ml Penicillin and 100U/ml Streptomycin (GE Healthcare).
18
2.2.1. Splitting of cells
In general, cells were grown to 80% confluency. To maintain optimal growth conditions the
cells were split in regular intervals. Therefore the medium was removed and cells were
washed with Phosphate buffered saline (PBS) (GE Healthcare). Cells were detached by
adding 1x Trypsin/EDTA (0.05%) (GE Healthcare) for approximately 2min at 37°C. The
reaction was stopped by adding standard medium. The cells were carefully resuspended and
1/8 to 1/10 of the cells were carefully transferred into a new tissue culture plate.
Table 3 shows the volumes of medium and Trypsin/EDTA used for the respective tissue
culture plates:
Table 3: Trypsin/EDTA volumes
Culture plates 10cm 6 well 24 well 96 well
Area (cm2) 58.95 9.6 2 0.36
Total volume 10ml 2ml 500μl 100μl
Trypsin/EDTA 1ml 250μl 100μl 50μl
2.2.2. Freezing of cells
The medium was removed and the cells were washed with PBS. The cells were detached by
adding Trypsin/EDTA (2.2.1) and centrifuged for 5min at 1000rpm (Beckman GS-6R swing-
out rotor). The supernatant was discarded and the cell pellet was resuspended in pre-cooled
freezing medium. Aliquots were transferred into pre-cooled cryotubes and were incubated for
30min on ice. The vials were stored at -80°C o/n and then transferred into liquid nitrogen or to
a freezer at -152°C for long term storage.
The freezing medium consisted of 70% standard medium (2.2), 20% FCS and 10% DMSO.
2.2.3. Thawing of cells
The cells were carefully thawed in a water bath at 37°C. As soon as the cells were thawed,
they were transferred to standard medium and then centrifuged for 5min at 1000rpm
19
(Beckman GS-6R swing-out rotor). After discarding the supernatant, the cell pellet was
resuspended in fresh standard medium and the cells were transferred onto tissue culture
plates.
2.2.4. Counting of cells
The cells were detached (3.2.1.) and appropriate dilutions were prepared. 10μl of the diluted
cell suspension was mixed with 10μl trypan blue solution 0.4% (Sigma) to exclude coloured
dead cells from counting. The cells were counted using a Neubauer-improved counting grid
and the number of cells was calculated according to the following formula:
mean value of four main squares x dilution factor x 10^4 = cells / ml
2.3. Transient transfection of cells
250μl serum-free Opti-MEM (Life Technologies) and TransIT®-LT1 transfection reagent
(Mirus) (3μl/μg DNA) were mixed and incubated at room temperature for 15min. 2-3μg
plasmid DNA were added followed by another 25min incubation at room temperature. The
TransIT®-LT1 reagent / DNA complexes were transferred to 6 well tissue culture plates and
5x10^5 cells were carefully seeded onto the transfection solution. After 18 to 24 hours the
cells were washed with PBS and standard medium was added.
2.4. Selection of stably transfected MEFs
In order to obtain stably transfected MEFs, the plasmid used for the transfection carried the
antibiotic resistance marker for puromycin and blasticidin respectively.
MEFs were transfected with the respective plasmid DNA as explained above (3.3). 24 hours
after replacing the transfection solution by fresh standard medium 2μg/ml puromycin and/or
3μg/ml blasticidin were added. The medium supplemented with the antibiotic was replaced
every three days. MEFs, which had not been transfected, served as controls.
After about 10 days of cultivation in the selective medium the growth of resistant clones was
observed. Cells from these clones were carefully detached by directly adding 2μl
Trypsin/EDTA to the cells and by gently pipetting. 1-3 cells from each clone were transferred
to 100μl standard medium supplemented with the respective antibiotic in 96 well tissue
20
culture plates. The selection process was continued for at least another 2 weeks and the cells
were expanded to 24 well and 6 well tissue culture plates, respectively to check protein
expression by Western Blot analysis.
2.5 Plasmids
The plasmids for the transgenic expression of the TYK2-Avitag fusion protein (7.1.) and of
the FLAG-tagged BirA enzyme (7.2.) were kindly provided by Dr.Ursula Reichart from the
Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna. For
details see the data sheets in the appendix.
2.6. Protein analysis
2.6.1. Lysing of cells
The cells were washed with ice-cold PBS and detached with an appropriate volume of
1xSchindler Lysis buffer (150μl/6well, 40μl/24 well) using a cell scraper. The lysate was
transferred into a tube and incubated on ice for 20min followed by a centrifugation at
16000rpm at 4°C for 5minutes. The supernatant was transferred into a new tube and stored at
-80°C.
1xSchindler Lysis buffer
NP40 0.5 %
TrisHCl pH 8 50 mM
NaCl 150 mM
Glycerol 10 %
DTT 2mM
EDTA 0.1 mM
Na-vanadate 0.2 mM
Na-fluoride 25 mM
PMSF 1 mM
21
2.6.2. Determination of the protein concentration
The concentration of proteins was determined by using a Bradford protein assay (BIO-RAD).
2μl of the lysate were mixed with the Bradford reagent and incubated at room temperature for
20min. The absorbance was measured with a spectrophotometer (Hitachi U-300) at 595nm.
The protein concentration was calculated using a BSA standard curve with a measurable
range from 1 – 10μg protein as reference.
2.6.3. SDS-polyacrylamide gel electrophoresis (PAGE)
SDS PAGE was used to separate proteins dependant on their molecular weight. SDS-
polyacrylamide gels consisted of a resolving gel (7.5 to 10% acrylamide/bis (375:1) (BIO-
RAD), 375 mM Tris/HCl pH 8.8, 0.1% SDS, 0.1% APS, 0.05% TEMED) for an optimal
separation of the molecules and a stacking gel (5% acrylamide/bis (37.5:1), 125mM Tris/HCl
pH 6.8, 0.1% SDS, 0.1% APS, 0.05% TEMED) for sharpening the bands by concentrating the
proteins. SDS running buffer (25mM Tris, 192mM glycine, 0.1% SDS) was used for the
electrophoresis.
The lysates were mixed with an equal volume of 2xLämmli sample buffer (LSB; 2% SDS
4%, 126mM Tris/HCl pH 6.8, 20% glycerol, 200mM DTT, 0.02% bromphenol blue)
incubated at 95°C for 5min and subsequently chilled on ice before loading the gel. A
prestained protein marker (PageRulerTM, Fermentas) was used as a molecular weight standard.
The gel electrophoresis was performed at 100 volt using SDS running buffer (25mM Tris,
192mM glycine, 0.1% SDS).
2.6.4. Western Blot analysis
After separating the proteins by SDS-PAGE the gels were blotted onto nitrocellulose
membranes (GE Healthcare) using 1x transfer buffer (25mM Tris/HCl, 150mM glycine, 20%
methanol). The transfer was performed either by a semy-dry blotter (Trans-Blot® SD (BIO-
RAD) at 100mA for 2hrs or by a wet tank system (Mini Trans-Blot® cell (BIO-RAD) at
350mA for 2hrs. When using the wet tank system steady conditions were ensured by constant
recirculation and cooling the transfer buffer.
22
In order to check transfer efficiency the blotted membrane was stained with Ponceau-S
solution (Sigma). After destaining the membranes with H2Odest. unspecific binding sites
were blocked by incubation in blocking solution (5% milk powder in PY-TBST) for 2hrs at
room temperature. After a washing step with PY-TBST (10mM Tris/HCl pH 7.4, 75mM
NaCl, 1mM EDTA pH8, 0.1% Tween20) the blots were incubated with the first antibody over
night at 4°C with constant agitation. On the next day, the blots were washed three times for
15min with PY-TBST and incubated with a horse radish peroxidase (HRP) conjugated second
antibody for 50-60min at room temperature. After repeating the washing step again for three
times, the membranes were probed using the ECL detection system (Amersham Biosciences).
Finally the chemiluminescent signals were detected after the exposure to a radiographic film.
Table 4: Antibodies used for Western blotting
antigen Antibody source Company concentration
TYK2 anti-TYK2 Rabbit piCHEM Forschungs- und Entwicklungs GmbH, Graz, Austria 1:2000
FLAG anti-ECS-bethyl-FLAG Goat Bethyl Laboratories, Inc.
Montgomery, USA 1:4000
STAT1 α/β anti-STAT1 α/β Rabbit Cell Signaling Technology, Leiden,
The Netherlands 1:2000
pSTAT1 anti-pSTAT1 α/β (Tyr701) Rabbit Cell Signaling Technology, Leiden,
The Netherlands 1:1000
NFκB p65 Anti- NFκB p65 Rabbit Miilipore, Bedford MA, USA 1:2000
rabbit IgG anti-rabbit IgG Goat Jackson ImmunoResearch, Suffolk,
UK 1:20000
goat IgG anti-goat IgG Rabbit Jackson ImmunoResearch, Suffolk,
UK 1:20000
23
2.6.4.1. Stripping of blotted membranes
For re-probing the blotted membranes were incubated in stripping buffer (2mM glycine/HCl
pH 2.5, 0.25% SDS) for 30min at room temperature or at 4°C for extended periods.
Afterwards, the blots were rinsed with hot tap water and PY-TBST followed by incubation in
1mM Tris/HCl pH8 for 15min at room temperature. After a short washing step with PY-
TBST the blots were ready for probing with another first antibody.
2.7. Purification of biotinylated TYK2
2.7.1. Separation of biotinylated protein
Streptavidin provides four sites for the binding of biotin residues. This binding reaction is
characterised by high efficiency and specificity. Neutravidin is a modified variant of the
streptavidin molecule displaying minimal nonspecific binding. For the purification of
biotinylated TYK2 protein NeutrAvidinTM Agarose Resin (ThermoScientific) was used
according to the manufacturer´s protocol.
After the equilibration of all reagents at room temperature 0.8ml centrifuge columns (Pierce)
were packed with 100μl of 50% neutravidin resin slurry and placed into collection tubes. The
columns were centrifuged for 1min then 500μl binding buffer TBS/NP40 (50mM Tris,
150mM NaCl, 0.3% NP40, pH 7.5) were added followed by another centrifugation of 1min to
remove all traces of the storage buffer. The washing step was repeated twice. All
centrifugation steps during the protocol were performed at 1000g and room temperature.
Subsequently, 100 or 200μg total protein lysate were added to the resin packed column
followed by an incubation for 1h at room temperature. Then the column was washed with
500μl binding buffer TBS/NP40 to separate resin bound biotinylated from non-biotinylated
proteins and the flow through was collected (Flow1). This step was repeated twice also
collecting the flow through of the second washing step (Flow2).
2.7.2. Elution of biotinylated protein
The resin was collected from the centrifuge columns and was transferred into a 1.5ml tube.
80μl 1xLämmli sample buffer were mixed with the resin by thorough vortexing. The resin
was boiled for 5min at 100°C and centrifuged for 1min at 1000xg and chilled on ice. The
24
supernatant containing the eluated biotinylated proteins was transferred into a new tube and
stored at -20°C.
2.7.3. Precipitation of proteins
The non-resin bound proteins collected with the flow through were concentrated by acetone
precipitation.
One volume of protein solution was mixed with one volume of acetone (pre-cooled at -20°C)
and incubated for at least 1h. The tubes were centrifuged at 15000xg at 4°C for 10min. the
supernatants were carefully discarded and the tubes were left uncapped for 30min to 1h at
room temperature to allow evaporation of residual acetone. Finally, the protein pellet was
resuspended in 50μl 1xLämmli sample buffer and stored at -20°C.
2.8. Proof of functionality of tagged TYK2
To prove the functionality of biotinylated TYK2, we checked tyrosine phosphorylation of
STAT1 following stimulation with interferon α.
TYK2 deficient U1A cells (2.1.3.) were transiently transfected (2.3.) to express either
Tyk2/AviTag or both Tyk2/AviTag and BirA/FLAG.
For the interferon treatment cells were counted and 5x10^5 cells were put into 6well tissue
culture plates. Directly before the experiment the cells were washed twice with PBS (GE
Healthcare) and fresh medium including 1000IU Universal Type I Interferon
(PBLInterferonSource) /ml medium was added.
After 30min incubation at 37°C cells were lysed and tested for phosphorylated STAT1 by
Western blot analysis. Untransfected U1A cells served as negative controls and 2fTGH cells
as positive wild-type controls.
25
2.9. List of materials and reagents
Table 5: Cell culture reagents Reagent Abbreviation Company Dulbecco´s Modified Eagle Medium DMEM GE Healthcare, Vienna, Austria
Phosphate-Buffered Saline PBS GE Healthcare, Vienna, Austria
Fetal Calf Serum FCS Life Technologies, Vienna, Austria
L-Glutamin 200mM (100x) L-GLU GE Healthcare, Vienna, Austria
Penicillin Streptomycin; 10000U/ml Penicillin + 10000μ/ml Streptomycin
Pen/Strep GE Healthcare, Vienna, Austria
β-Mercaptoethanol β-Mer Life Technologies, Vienna, Austria
Trypsin-EDTA 0,05% Trypsin GE Healthcare, Vienna, Austria
Opti-MEM I Reduced Serum Medium Opti-MEM Life Technologies, Vienna, Austria
RPMI 1640 medium (with L-Glutamine)
RPMI GE Healthcare, Vienna, Austria
TransIT-LT1 Transfection Reagent / Mirus Bio LLC, Wisconsin, USA
Materials:
Falcon TM cell culture dishes BD Bioscience Austria (Schwechat, Austria)
Centrifuge adaptors and tubes Beckmann (Krefeld, Germany)
Cryotubes Greiner (Kremsmünster, Austria)
Electrophoresis Chamber, Buffers and
Detergents
Bio-Rad Laboratories Gmbh (Wien,
Austria)
26
3. Results
3.1. Generation of MEFs stably co-expressing Tyk2/AviTag and BirA/FLAG
The AviTag/BirA system comprises two different components. The transgenic construct
pTyk2/AviTag-puro provides the TYK2 cDNA c-terminally tagged with the AviTag
sequence, which serves as the biotinylatin site (see 1.6). The second transgenic construct
pBirA/FLAG-blast contains the cDNA of the bacterial biotin ligase BirA. The BirA construct
is C-terminally tagged with a FLAG tag to facilitate the specific detection of the BirA protein
(2.6).
Only if both proteins, the biotinylation site carrying TYK2 as well as the biotin ligase are
expressed simultaneously, the AviTag/BirA system is functionally active resulting in the
efficient biotinylation of the tagged protein of interest.
MEFs stably co-expressing Tyk2/AviTag and BirA/FLAG were generated by two approaches,
serial transfection and co-transfection of the constructs, respectively. Note, that due to the
varying quality of the commercially available anti-TYK2 antibodies in Western blot
experiments cross-reacting protein(s) may occur; their appearance is not discussed
specifically in each figure.
3.1.1. Serial transfection of the AviTag/BirA constructs
At first, TYK2 deficient MEFs were transfected with pTyk2/AviTag-puro. Putative positive
clones were selected for several weeks with 2μg/ml puromycin and the TYK2 expression was
screened by Western Blot analysis.
Positive clones showed a signal at approximately 130kDa (Fig.1A).
Fig.1A: Western blot analysis of TYK2 expression in different MEF clones 8 weeks after transfection. Clones A72, A74, A75, G92 and G93 show adequate expression of TYK2.
α-TYK2 - 130 kDa
A72 A74 A75 G91 G92 G93
27
Subsequently, positive clones for Tyk2/AviTag-puro were transfected with pBirA/FLAG-
blast and double positive clones were selected with 3μg/ml blasticidin and 2μg/ml puromycin.
After another several weeks of antibiotic selection the resistant clones were screened by
Western Blot analysis detecting the FLAG-tagged BirA protein at the expected site of
approximately 35kDa (Fig.1B).
The clones A75, G92 and G93 showed stable expression of both proteins, Tyk2/AviTag and BirA/FLAG.
3.1.2. Co-Transfection of Tyk2/AviTag and BirA/FLAG
In the next set of experiments, TYK2 deficient MEFs were transfected with pTyk2/AviTag-
puro and pBirA/FLAG simultaneously in a single reaction. Clones were double selected with
2μg/ml puromycin and 3μg/ml blasticidin for several weeks. In putative positive antibiotic
resistant clones the expression of Tyk2/AviTag and BirA/FLAG was checked by Western
Blot analysis.
Fig.2: Western blot analysis of TYK2 and BirA/ expression in different clones 8 weeks after transfection. Clones positive for TYK2 show a signal at 130 kDa. Clones positive for BirA show a signal at 35kDa. Only clone Y8 expresses both constructs.
α-BirA/FLAG - 35 kDa
Y3 Y4 Y5 Y7 Y8 Y10
- 130 kDa α-TYK2
Fig.1B: Western blot analysis of BirA expression in different serially transfected MEF clones 8 weeks after transfection. Clones A75, G92 and G93 show distinct BirA expression.
α-BirA/FLAG - 35 kDa
A72 A74 A75 G91 G92 G93
α-BirA/FLAG - 35 kDa
A72 A74 A75 G91 G92 G93
28
The co-transfection approach revealed only one clone (Y8) stably expressing both proteins
Tyk2/AviTag and BirA/FLAG.
3.1.3. Validation of the stable genome integration of Tyk2/AviTag and BirA/FLAG
To ensure the stable integration of Tyk2/AviTag and BirA/FLAG into the genome of the
transfectants, the selected positive clones (A75, G92, G93, Y8) were passaged again for
several times under selective culture conditions. With the exception of clone G93 where the
stable integration of both constructs failed, the residual clones A75, G92 and Y8 showed
stable and efficient expression of the transgenic proteins Tyk2/AviTag and BirA/FLAG
(Fig.3A+B).
Subsequently the clones were subdivided into 6 different wells each and passaged twice
before being screened by Western blotting. All three remaining clones expressed
Tyk2/AviTag and BirA/FLAG in adequate amounts (Fig.3B).
A75 G92 G93 Y8 A75 G92 G93 Y8
Fig.3A: Screening for Tyk2/AviTag and BirA/FLAG expression to ensure stable genome integration. Clones A75, G92 and Y8 show efficient expression of both constructs.
α-Tyk2
α-BirA/FLAG
- 130 kDa
- 35 kDa
29
3.2. Purification of neutravidin-bound biotinylated TYK2 protein
The high affinity of biotin for avidin and streptavidin is an established and reliable method for
the purification of biotinylated proteins. In mammalian cells only very few proteins get
biotinylated, so there is a minimal chance for cross-reactions. Thus, the detection of
artificially biotinylated protein by streptavidin binding methods is facilitated.
A751 A752 A753 A754 A755 A756
- 35 kDa
- 130 kDa α-TYK2
α-BirA/FLAG
- 35 kDa
- 130 kDa
G921 G922 G923 G924 G925 G926
α-TYK2
α-BirA/FLAG
- 130 kDa
Y81 Y82 Y83 Y84 Y85 Y86
α-TYK2
α-BirA/FLAG - 35 kDa
A751 A752 A753 A754 A755 A756
- 35 kDa
- 130 kDaα-TYK2
α-BirA/FLAG
- 35 kDa
- 130 kDa
G921 G922 G923 G924 G925 G926
α-TYK2
α-BirA/FLAG
- 130 kDa
Y81 Y82 Y83 Y84 Y85 Y86
α-TYK2
α-BirA/FLAG - 35 kDa
Fig.3B: The clones A75, G92 and Y8 were subdivided and screened for Tyk2/AviTag and BirA/FLAG by Western Blot analysis. All of them show a stable expression of both constructs.
30
The purpose was to show that Tyk2/AviTag gets efficiently biotinylated and can be purified
by binding to neutravidin beads.
The specific biotinylation by BirA without the contribution of the endogenous mammalian
binding ligase had to be revealed.
To prove the functionality of the AviTag/BirA biotinylation system transiently co-transfected
TYK2 deficient MEF cells were used. Before validating the biotinylation of TYK2, the
expression of both AviTag/BirA system components, Tyk2/AviTag and BirA/FLAG, was
confirmed (Fig.4A).
The proteins of transfected MEFs and non-transfected controls were lysed and biotinylated
TYK2 protein was immobilised onto neutravidin beads. The beads were cleared from
unbound proteins by several washing steps. Both fractions were checked by Western blot
analysis, biotinylated proteins eluated from the beads (E) and unbound protein precipitated
from the flow through (F).
Fig.4A: Western blot analysis of TYK2 and BirA/FLAG expression in transiently transfected MEF cells. T/B: Cells were transfected with both Tyk2/AviTag and BirA/FLAG; T: Cells were transfected with Tyk2/AviTag only; w/o: non-transfected Tyk2-/- MEFs were used as a control.
w/o T T/B
- 35 kDa
- 130 kDa
α-BirA/FLAG
α-TYK2
31
The Western blot analysis revealed the successful biotinylation of TYK2. The biotinylation
occurs exclusively in the presence of BirA. The endogenous biotin ligase is not able to
biotinylate the AviTag. Non-transfected cells only show an unspecific band above the TYK2
signal (Fig.4B).
Since MEFs usually show transfection rates below 50% when lipofection is used only small
amounts of biotinylated TYK2 were observed.
When MEFs stably expressing Tyk2/AviTag and BirA were used the amounts of biotinylated
TYK2 clearly excelled the amounts of unbound TYK2 in the flow throughs (F) (Fig.4C).
Fig.4B: Western blot analysis of bound (E) and unbound (F1 – first flow through) protein after purification by neutravidin. T/B: Cells were transfected with both Tyk2/AviTag and BirA/FLAG; T: Cells were transfected with Tyk2/AviTag only; w/o: non-transfected Tyk2-/- MEFs were used as a control. Biotinylation of TYK2 occurs only in the presence of BirA.
E F1 E F1 E F1
- 130 kDa α-TYK2
T/B T w/o
E F1 E F1 E F1
- 130 kDaα-TYK2
T/B T w/o
Fig.4C: Western blot analysis of bound (E) and unbound (F1 – first flow through) protein after purification by neutravidin. MEF clones stably expressing Tyk2/AviTag and BirA/FLAG were used (A75, G92 and Y8). Untransfected MEF TYK2-/- cells were used as control (w/o).
E F1 E F1 E F1 E F1
- 130 kDa
A75 G92 Y8 w/o
α-TYK2
32
3.3. Validation of the Tyk2/AviTag protein
There is the possibility that signalling through TYK2 is changed, limited or even lost because
of the C-terminally tagged biotinylation site or the changes in the protein structure by the
biotinylation itself.
The signal transducer and activator of transcription (STAT)1 is a transcription factor which
gets phosphorylated by TYK2 upon binding of interferon α to the receptor. To test the
functionality of the tagged TYK2 the components of the AviTag/BirA system were transiently
overexpressed in U1A cells. These cells are TYK2 deficient and therefore unresponsive to
IFNα (see 2.8). 2fTGH cells, expressing wild-type TYK2 served as control (Fig.5A).
TYK2 signalling was fully reconstituted in U1A cells by the expression of Tyk2/AviTag
resulting in the phosphorylation of STAT1 upon IFNα stimulation (Fig.5B).
T T T/B T/B w/o w/o
- 35 kDa
α-TYK2
α-BirA/FLAG
- 130 kDa
Fig.5A: Western blot analysis of U1A cells transiently transfected with Tyk2/AviTag (T) or co-transfected with Tyk2/AviTag and BirA/FLAG (T/B). Non-transfected cells (w/o) served as control.
33
An efficient phosphorylation of STAT1 upon IFNα stimulation was observed when
biotinylated TYK2 protein was expressed. Thus, neither the peptide tag itself nor the
biotinylation site interfered with the signalling properties of TYK2.
However, compared to wild-type 2fTGH cells the level of phosphorylated STAT1 appears to
be lower in TYK2 reconstituted U1A cells. The reasons for this are discussed.
U1A 2fTGH
T T T/B T/B w/o w/o
STAT1 α/β
p-STAT1
p65NFKB
0´ 30´ 0´ 30´ 0´ 30´ 0´ 30´ IFN α
- 100 kDa
- 100 kDa
- 70 kDa
U1A 2fTGH
T T T/B T/B w/o w/o
STAT1 α/β
p-STAT1
p65NFKB
0´ 30´ 0´ 30´ 0´ 30´ 0´ 30´ IFN α
- 100 kDa
- 100 kDa
- 70 kDa
Fig.5B: Phosphorylation of STAT1 after IFNα stimulation of U1A cells transfected with Tyk2/AviTag (T) or co-transfected with Tyk2/AviTag and BirA/FLAG (T/B). Non-transfected U1A cells (w/o) and wild-type responsive 2fTGH cells served as controls.
34
4. Discussion
Tyrosine kinase (TYK) 2 is a member of the Janus kinase family. As a key player in the
JAK/STAT signalling pathway it plays an important role in cytokine signalling.
In general TYK2 is attributed a predominantly protective role in infectious and a mainly
deleterious role in inflammatory diseases (Strobl et al. 2011 and refs therein). In addition
TYK2 was described to have an impact for example in neuropathological disorders (Wan et
al. 2010), in cancer pathogenesis (Ide et al. 2008, Song et al. 2008, Zhu et al. 2009), in
lymphoid tumours (Stoiber et al. 2004) and in autoimmune diseases like experimental
encephalomyelitis and arthritis (Oyamada et al. 2009).
By now, two human TYK2 deficient patients were reported, suffering from a variety of
disorders (Minegishi et al. 2006, Kilic et al. 2012), genome wide association studies and
targeted next generation sequencing approaches have identified TYK2 polymorphism in
human metabolic and autoimmune diseases (e.g. Diogo et al.2015, Wallace et al. 2010) and
TYK2 gene fusions have been identified in human haematopoietic cancers (Crescenzo et al.
2015, Roberts et al. 2014, Velusamy et al. 2014).
The inhibition of TYK2 seems to be a promising tool for example in the treatment of psoriasis
and inflammatory bowel disease (IBD).
Until now, only a few TYK2 interaction partners like IFNAR1 (Richter et al. 1998),
suppressor of cytokine signalling (SOCS)-3 (Zeng et al. 2008), SHP2 (Schaper et al. 1998) or
CD36 (Venugopal et al. 2004) were confirmed by conventional techniques like
immunoprecipitation. The precise mechanisms how TYK2 contributes to canonical or non-
canonical signalling remain often still unclear due to lacking knowledge about molecule
interactions.
These findings in basic and translational research lead to the necessity to identify further
interaction partners of TYK2.
In this study, the tagging system AviTag/BirA was used to get a better insight into the
usability of this system for detecting interaction partners of TYK2.
35
Conventional tagging systems are mainly developed and optimised for high-throughput
screenings and mostly use bacterial or in vitro cell culture expression systems. In contrast, the
AviTag/BirA tagging system is engineered for tagging proteins of interest in vivo (de Boer et
al. 2003). In this way, it might be an ideal system to detect molecules interacting with TYK2
during experimental challenges leading to TYK2 dependent diseases.
The current study shows the functionality, efficiency and specificity of the AviTag/BirA
system in TYK2 deficient murine fibroblasts. Compared to transiently transfected cells, cells
with stable integration of the tagging system components showed a markedly higher yield of
biotinylated TYK2. This underlines that stable genome integration should be used for the
efficient purification of the protein of interest and potential interacting molecules.
In addition, the functionality of the tagged TYK2 protein was confirmed by the successful
reconstitution of STAT1 phosphorylation after stimulation with interferon alpha in TYK2
deficient cells. However, compared to wild-type control cells a reduced phosphorylation
signal was observed.
Future investigations should clarify whether transfected U1A cells or wild-type 2fTGH cells
express equal amounts of total STAT1 and/or TYK2 protein. If effector protein differences
are not the cause of the reduced STAT1 phosphorylation, specific investigations should
clarify if the functionality of the tagged TYK2 is generally impaired or altered. This could be
done e.g. by in vivo kinase assays.
Emphasising the applicability of the AviTag/BirA system for mammalian in vivo systems, this
study provides a solid base for subsequent approaches like a knock-in mouse model by
introducing the tag sequence into the endogenous TYK2 gene locus.
In conclusion, in this study the AviTag/BirA system was successfully introduced into TYK2
deficient primary cells and thus will support further approaches to identify interaction partners
of TYK2 by in vivo tagging.
36
5. Summary
Tyrosine kinase (TYK) 2, a member of the family of Janus kinases, takes part in a number of
different signalling pathways. As a receptor kinase TYK2 is involved in inflammation events,
allergy, autoimmune disease and cancer.
So far, only very few direct interaction partners of TYK2 were already verified by evidence.
The aim of this study was to establish prerequisites for finding further interaction partners of
TYK2. Therefore a novel tagging system, the AviTag/BirA system, was applied. It is based
on the biotinylation of an artificial biotinylation site (AviTag) fused to the protein of interest.
It requires the simultaneous expression of the tagged protein of interest and the biotin protein
ligase BirA.
In this study TYK2 deficient murine embryonal fibroblasts were either serially or
simultaneously transfected with both components of the tagging system, namely the fusion
protein Tyk2/AviTag and BirA/FLAG. Three independent cell clones were established which
showed efficient and stable expression of biotinylated TYK2.
By purification via neutravidin resin specifically biotinylated TYK2 protein was recovered.
As affinity tags might have an effect on the protein they are fused to it was decided to check if
signalling through TYK2 is altered by the tag itself or by the attachment of the biotin
molecule.
Therefore, the phosphorylation of STAT1 as a consequence of stimulation with interferon
alpha was checked in the TYK2 deficient cell line U1A. TYK2 signalling was reconstituted
when Tyk2/AviTag and BirA/FLAG were simultaneously overexpressed leading to
phosphorylation of the transcription factor STAT1.
In summary, we report the successful generation of primary mammalian cells stably
expressing the AviTag/BirA tagging system. The functionality of the AviTag/BirA system
was demonstrated and resulted in the efficient and specific biotinylation of kinase active
TYK2. Thus, biotinylated TYK2 can serve as a valuable model to investigate interaction
partners of TYK2.
37
5.1 Zusammenfassung
Tyrosinkinase (TYK) 2, ein Mitglied der Familie der Januskinasen, ist ein wichtiger
Bestandteil einer Reihe von Signaltransduktionswegen, die sich im Rahmen von
Entzündungsgeschehen, allergischen Reaktionen, Autoimmunerkrankungen und
neoplastischen Erkrankungen als wichtig erwiesen haben.
Bis zum jetzigen Zeitpunkt konnten nur sehr wenige direkte Interaktionspartner von TYK2
nachgewiesen werden.
Das Ziel dieser Arbeit bestand darin, durch ein neuartiges Tagging-System, das sogenannte
AviTag/BirA System, den Weg zur Auffindung weiterer Interaktionspartner von TYK2 zu
ebnen. Das AviTag/BirA System basiert auf der Biotinylierung einer künstlichen
Biotinylierungsstelle, die dem Ziel-Protein hinzugefügt wird. Um eine erfolgreiche
Biotinylierung zu ermöglichen ist die simultane Expression des getaggten Zielproteins und
der Biotin-Protein-Ligase BirA zu gewährleisten.
Es wurden auf zwei unterschiedlichen Wegen – durch simultane oder serielle Transfektion -
murine embryonale Fibroblasten erzeugt, welche die beiden notwendigen Elemente des
AviTag-Systems – AviTag getagtes TYK2 und BirA - stabil und in ausreichenden Mengen
exprimieren.
Das durch BirA spezifisch biotinylierte TYK2 wurde über Neutravidin Säulen aufgereinigt.
In weiterer Folge wurde überprüft, ob die Biotinylierung des Proteins oder das Tagging per se
die Funktionalität von TYK2 negativ beeinflussen. Zu diesem Zweck wurde die TYK2
defiziente Zelllinie U1A mit den Komponenten des Tagging Systems, Tyk2/AviTag und
BirA/FLAG, transfiziert und anschließend mit Interferon alpha stimuliert. Die daraus
resultierende Phosphorylierung des Transkriptionsfaktors STAT1 zeigte die Funktionalität
von biotinyliertem TYK2 als Rezeptorkinase.
Zusammenfassend wird die erfolgreiche Generierung von Zellen beschrieben, die konstant
Tyk2/AviTag und BirA/FLAG exprimieren und dadurch die Aufreinigung von biotinyliertem
TYK2 ermöglichen. Da dessen Funktionalität erhalten ist, kann biotinyliertes TYK2 einen
wertvollen Beitrag leisten um Interaktionspartner von TYK2 zu erforschen.
38
6. References
Aaronson DS, Horvath CM. 2002. A road map for those who don't know JAK-STAT.
Science, 296(5573):1653-5.
Arnau J, Lauritzen C, Petersen GE, Pedersen J. 2005. Current strategies for the use of affinity
tags and tag removal for the purification of recombinant proteins. Protein Expr Purif, 48(1):1-
13
Beckett D, Kovaleva E, Schatz PJ. 1999. A minimal peptide substrate in biotin holoenzyme
synthetase-catalyzed biotinylation. Protein Sci, 8(4):921-9
Bidwell GL 3rd, Raucher D. 2005. Application of thermally responsive polypeptides directed
against c-Myc transcriptional function for cancer therapy. Mol Cancer Ther. 4(7):1076-85
Bieber AJ, Suwansrinon K, Kerkvliet J, Zhang W, Pease LR, Rodriguez M. 2010. Allelic
variation in the Tyk2 and EGF genes as potential genetic determinants of CNS repair. Proc
Natl Acad Sci U S A, 107(2):792-7
Blumenthal DK, Takio K, Edelman AM, Charbonneau H, Titani K, Walsh KA, Krebs EG.
1985. Identification of the calmodulin-binding domain of skeletal muscle myosin light chain
kinase. Proc Natl Acad Sci U S A, 82(10):3187-91
Bogdan C, Mattner J, Schleicher U. 2004. The role of type I interferons in non viral infections
Immunol Rev, 202:33-48
Bucher MH, Evdokimov AG, Waugh DS. 2002. Differential effects of short affinity tags on
the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallogr D
Biol Crystallogr, 58(Pt 3):392-7
39
Busso D, Delagoutte-Busso B, Moras D. 2005. Construction of a set Gateway-based
destination vectors for high-throughput cloning and expression screening in Escherichia coli.
Anal Biochem, 343(2):313-21
Chant A, Kraemer-Pecore CM, Watkin R, Kneale GG. 2005. Attachment of a histidine tag to
the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA
causes a conformational change at the DNA-binding site. Protein Expr Purif, 39(2):152-9
Chen H, Xu Z, Xu N, Cen P. 2005. Efficient production of a soluble fusion protein containing
human beta-defensin-2 in E. coli cell-free system. J Biotechnol, 115(3):307-15
Chapman-Smith A, Cronan JE Jr. 1999. The enzymatic biotinylation of proteins: a post-
translational modification of exceptional specificity. Trends Biochem Sci, 24(9):359-63
Chapman-Smith A, Cronan JE Jr. 1999. Molecular biology of biotin attachment to proteins. J
Nutr, 129(2S Suppl):477S-484S
Crescenzo R, Abate F, Lasorsa E, Tabbo´F, Gaudiano M, Chiesa N, Di giacomo F,
Spaccarotella E, Barbarossa L, Ercole E, Todaro M, Boi M, Acquaviva A, Ficarra E, Novero
D, Rinaldi A, Tousseyn T, Rosenwald A, Kenner L, Cerroni L, Tzankov A, Ponzoni M, Paulli
M, Weisenburger D, Chan WC, Iqbal J, Piris MA, Zamo´A, Ciardullo C, Rossi D, Gaidano G,
Pileri S, Tiacci E, Falini B, Shultz LD, Mevellec L, Vialard JE, Piva R, Bertoni F, Rabadan R,
Inghirami G; European T-Cell Lymphoma Study Group, T-Cell Project: Prospective
Collection of Data in Patients with Peripheral T-Cell Lymphoma and the AIRC 5xMille
Consortium “Genetics-Driven Targeted Management of Lymphoid Malignancies”. 2015.
Convergent Mutations and Kinase Fusions Lead to Oncogenic STAT3 Activation in
Anaplastic Large Cell Lymphoma. Cancer Cell, 27(4):516-32
40
de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E, Heck A, Grosveld F,
Strouboulis J. 2003. Efficient biotinylation and single-step purification of tagged transcription
factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A, 100(13):7480-5
de Vries E.G.E., M.N. de Hooge, J.A. Gietema, S. de Jong, Correspondencere: C.G. Ferreira
et al. 2003. Apoptosis: Target of Cancer Therapy. Clin. Cancer Res, 9 (2003) 912
Decker T, Müller M, Stockinger S. 2005. The yin and yang of type I interferon activity in
bacterial infection. Nat Rev Immunol, 5(9):675-87
Diogo D, Bastarache L, Liao KP, Graham RR, Fulton RS, Greenberg JD, Eyre S, Bowes J,
Cui J, Lee A, Pappas DA, Kremer JM, Barton A, Coenen MJ, Franke B, Kiemeney LA,
Mariette X, Richard-Miceli C, Canhão H, Fonseca JE, de Vries N, Tak PP, Crusius JB,
Nurmohamed MT, Kurreeman F, Mikuls TR, Okada Y, Stahl EA, Larson DE, Deluca TL,
O´Laughlin M, Fronick CC, Fulton LL, Kosoy R, Ransom M, Bhangale TR, Ortmann W,
Cagan A, Gainer V, Karlson EW, Kohane I, Murphy SN, Martin J, Zhernakova A, Klareskog
L, Padyukov L, Worthington J, Mardis ER, Seldin MF, Gregersen PK, Behrens T,
Raychaudhuri S, Denny JC, Plenge RM. 2015. TYK2 Protein-Coding Variants Protect against
Rheumatoid Arthritis and Autoimmunity, with No Evidence of Major Pleiotropic Effects on
Non-Autoimmune Complex Traits. PLoS One, 10(4):e0122271
Donnelly R, Dickensheets H, Finbloom D. 1999. The interleukin-10 signal transduction
pathway and regulation of gene expression in mononuclear phagocytes. Journal of Interferon
and Cytokine Research, 19:563–573
Dornhoff H, Siebler J, Neurath MF. 2011. Potential role of interferon-lambda in the treatment
of inflammation and cancer: an update. International Journal of Interferon, Cytokine and
Mediator Research, 3 51–57
41
Driegen S, Ferreira R, van Zon A, Strouboulis J, Jaegle M, Grosveld F, Philipsen S, Meijer D.
2005. A generic tool for biotinylation of tagged proteins in transgenic mice. Transgenic Res,
14(4):477-82.
Dudakov JA, Hanash AM, van den Brink MR. 2015. Interleukin-22: Immunobiology and
pathology. Annu Rev Immunol, 33:747-85
Goel A, Colcher D, Koo JS, Booth BJ, Pavlinkova G, Batra SK. 2000. Relative position of the
hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.
Biochim Biophys Acta, 1523(1):13-20
Head JF. 1992. A better grip on calmodulin. Curr Biol, 2(11):609-11
Hiller Y, Bayer EA, Wilchek M. 1990. Nonglycosylated avidin. Methods Enzymol, 184:68-
70.
Ide H, Nakagawa T, Terado Y, Kamiyama Y, Muto S, Horie S. 2008. Tyk2 expression and its
signaling enhances the invasiveness of prostate cancer cells. Biochem Biophys Res Commun,
369(2):292-6.
Kapust RB, Waugh DS. 1999. Escherichia coli maltose-binding protein is uncommonly
effective at promoting the solubility of polypeptides to which it is fused. Protein Sci,
8(8):1668-74.
Karaghiosoff M, Neubauer H, Lassnig C, Kovarik P, Schindler H, Pircher H, McCoy B,
Bogdan C, Decker T, Brem G, Pfeffer K, Müller M. 2000. Partial impairment of cytokine
responses in Tyk2-deficient mice. Immunity, 13(4):549-60.
Kay BK, Thai S, Volgina VV. 2009. High-throughput biotinylation of proteins. Methods Mol
Biol, 498:185-96
42
Kilic SS, Hacimustafaoglu M, Boisson-Dupuis S, Kreins AY, Grant AV, Abel L, Casanova JL. 2012. A patient with tyrosine kinase 2 deficiency without hyper-IgE syndrome. J Pediatr, 160(6):1055-7
Kim KM, Yi EC, Baker D, Zhang KY. 2001. Post-translational modification of the N-
terminal His tag interferes with the crystallization of the wild-type and mutant SH3 domains
from chicken src tyrosine kinase. Acta Crystallogr D Biol Crystallogr, 57(Pt 5):759-62
Kotenko S. 2011. IFN-λs. Current Opinion in Immunology 23:583-590
Lane MD, Rominger KL, Young DL, Lynen F. 1964. The enzymatic synthesis of
holotranscarboxylase from apotranscarboxylse and (+) biotin. Investigation of the reaction
mechanism, 239:2865-71
Levy DE, Darnell JE. 2002. Stats: transcriptional control and biological impact. Nat Rev Mol
Cell Biol, 3(9):651-62
Liang J, Tsui V, Van Abbema A, Bao L, Barrett K, Beresini M, Berezhkovskiy L, Blair WS,
Chang C, Driscoll J, Eigenbrot C, Ghilardi N, Gibbons P, Halladay J,Johnson A, Kohli PB,
Lai Y, Liimatta M, Mantik P, Menghrajani K, Murray J, Sambrone A, Xiao Y, Shia S, Shin
Y, Smith J, Sohn S, Stanley M, Ultsch M, Zhang B,Wu LC, Magnuson S. 2013. Lead
identification of novel and selective TYK2 inhibitors. Eur J Med Chem, 67:175-87
Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. 2005. Comparison of
affinity tags for protein purification. Protein Expr Purif, 41(1):98-105
Mayer A, Sharma SK, Tolner B, Minton NP, Purdy D, Amlot P, Tharakan G, Begent RH,
Chester KA. 2004. Modifying an immunogenic epitope on a therapeutic protein: a step
towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT). Br J
Cancer, 90(12):2402-10
43
Meyer DE, Kong GA, Dewhirst MW, Zalutsky MR, Chilkoti A. 2001. Targeting a genetically
engineered elastin-like polypeptide to solid tumors by local hyperthermia. Cancer Res,
61(4):1548-54
Minegishi Y, Saito M, Morio T, Watanabe K, Agematsu K, Tsuchiya S, Takada H, Hara T,
Kawamura N, Ariga T, Kaneko H, Kondo N, Tsuge I, Yachie A, Sakiyama Y, Iwata T,
Bessho F, Ohishi T, Joh K, Imai K, Kogawa K, Shinohara M, Fujieda M, Wakiguchi H, Pasic
S, Abinun M, Ochs HD, Renner ED, Jansson A, Belohradsky BH, Metin A, Shimizu N,
Mizutani S, Miyawaki T, Nonoyama S, Karasuyama H. 2006. Human tyrosine kinase 2
deficiency reveals its requisite roles in multiple cytokine signals involved in innate and
acquired immunity. Immunity, 25(5):745-55
Nakamura R, Shibata K, Yamada H, Shimoda K, Nakayama K, Yoshikai Y. 2008. Tyk2-
signaling plays an important role in host defense against Escherichia coli through IL-23-
induced IL-17 production by gammadelta T cells. J Immunol, 181(3):2071-5
O´Shea J, Schwartz D, Villarino A, Gadina M, McInnes I, Laurence A. 2015. The JAK-STAT
pathway: Impact on human disease and therapeutic intervention. Annu.Rev.Med. 66:311-28
Oyamada A, Ikebe H, Itsumi M, Saiwai H, Okada S, Shimoda K, Iwakura Y, Nakayama KI,
Iwamoto Y, Yoshikai Y, Yamada H. 2009. Tyrosine kinase 2 plays critical roles in the
pathogenic CD4 T cell responses for the development of experimental autoimmune
encephalomyelitis. J Immunol, 183(11):7539-46
Pellegrini S, Dusanter-Fourt I. 1997. The structure, regulation and function of the Janus
kinases (JAKs) and the signal transducers and activators of transcription (STATs). Eur J
Biochem, 248(3):615-33
Pellegrini S, John J, Shearer M, Kerr IM, Stark GR. 1989. Use of a selectable marker
regulated by alpha interferon to obtain mutations in the signaling pathway. Mol Cell Biol,
9(11):4605-12
44
Pendini NR, Bailey LM, Booker GW, Wilce MC, Wallace JC, Polyak SW. 2008. Microbial
biotin protein ligases aid in understanding holocarboxylase synthetase deficiency. Biochim
Biophys Acta, 1784(7-8):973-82
Porath J. 1992. Immobilized metal ion affinity chromatography. Protein Expr Purif, 3(4):263-
81
Prchal-Murphy M, Semper C, Lassnig C, Wallner B, Gausterer C, Teppner-Klymiuk I,
Kobolak J, Müller S, Kolbe T, Karaghiosoff M, Dinnyés A, Rülicke T, Leitner NR, Strobl B,
Müller M. 2012. TYK2 kinase activity is required for functional type I interferon responses in
vivo. PLoS One, 7(6):e39141
Ragimbeau J, Dondi E, Alcover A, Eid P, Uzé G, Pellegrini S. 2003. The tyrosine kinase
Tyk2 controls IFNAR1 cell surface expression. EMBO J, 22(3):537-47
Rajan SS, Lackland H, Stein S, Denhardt DT. 1998. Presesnce of an N-terminal polyhistidine
tag facilitates stable expression of an otherwise unstable N-terminal domain of mouse tissue
inhibitor of metalloproteinase-1 in Escherichia coli. Protein Expr Purif, 13(1):67-72
Rane SG, Reddy EP. 2002. JAKs, STATs and Src kinases in hematopoiesis. Oncogene,
21(21):3334-3358
Rani MR, Leaman DW, Han Y, Leung S, Croze E, Fish EN, Wolfman A, Ransohoff RM.
1999. Catalytically active TYK2 is essential for interferon-beta-mediated phosphorylation of
STAT3 and interferon-alpha receptor-1 (IFNAR-1) but not for activation of phosphoinositol
3-kinase. J Biol Chem, 274(45):32507-11
45
Rawlings JS, Rosler KM, Harrison DA. 2004. The JAK/STAT signalling pathway. J Cell Sci,
117(Pt 8):1281-3
Richter MF, Duménil G, Uzé G, Fellous M, Pellegrini S. 1998. Specific contribution of Tyk2
JH regions to the binding and the expression of the interferon alpha/beta receptor component
IFNAR1. J Biol Chem, 273(38):24723-9
Riley JK, Takeda K, Akira S, Schreiber RD 1999. Interleukin-10 receptor signaling through
the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-
inflammatory action. J Biol Chem, 274(23):16513-21
Roberts KG, Li Y, Payne-Turner D, Harvey RC, Yang YL, Pei D, McCastlain K, Ding L, Lu
C, Song G, Ma J, Becksfort J, Rusch M, Chen SC, Easton J, Cheng J, Boggs K, Santiago-
Morales N, Iacobucci I, Fulton RS, Wen J, Valentine M, Cheng C, Paugh SW, Devidas M,
Chen IM, Reshmi S, Smith A, Hedlund E, Gupta P, Nagahawatte P, Wu G, Chen X, Yergeau
D, Vadodaria B, Mulder H, Winick NJ, Larsen EC, Carroll WL, Heerema NA, Carroll AJ,
Grayson G, Tasian SK, Moore AS, Keller F, Frei-Jones M, Whitlock JA, Raetz EA, White
DL, Hughes TP, Guidry Auvil JM, Smith MA, Marcucci G, Bloomfield CD, Mrózek K,
Kohlschmidt J, Stock W, Kornblau SM, Konopleva M, Paietta E, Pui CH, Jeha S, Relling
MV, Evans WE, Gerhard DS, Gastier-Foster JM, Mardis E, Wilson RK, Loh ML, Downing
JR, Hunger SP, Willman CL, Zhang J, Mullighan CG. 2014. Targetable kinase-activating
lesions in Ph-like acute lymphoblastic leukemia. N Engl J Med, 371(11):1005-15
Rybak JN, Scheurer SB, Neri D, Elia G. 2004. Purification of biotinylated proteins on
streptavidin resin: a protocol for quantitative elution. Proteomics, 4(8):2296-9
Schaper F, Gendo C, Eck M, Schmitz J, Grimm C, Anhuf D, Kerr IM, Heinrich PC. 1998.
Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing
receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein
expression. Biochem J, 335( Pt 3):557-65
46
Schatz PJ. 1993. Use of peptide libraries to map the substrate specificity of a peptide-
modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli.
Biotechnology, 11(10):1138-43
Shaw MH, Boyartchuk V, Wong S, Karaghiosoff M, Ragimbeau J, Pellegrini S, Muller M,
Dietrich WF, Yap GS. 2003. A natural mutation in the Tyk2 pseudokinase domain underlies
altered susceptibility of B10.Q/J mice to infection and autoimmunity. Proc Natl Acad Sci U S
A, 100(20):11594-9
Shih, Y.P., Kung, W.M., Chen, J.C., Yeh, C.H., Wang, A.H.J., and Wang, T.F. 2002. High-
throughput screening of soluble recombinant proteins. Protein Sci, 11 1714–1719
Song XC, Fu G, Yang X, Jiang Z, Wang Y, Zhou GW 2008. Protein expression profiling of
breast cancer cells by dissociable antibody microarray (DAMA) staining. Mol Cell
Proteomics, 1:163-9
Stoiber D, Kovacic B, Schuster C, Schellack C, Karaghiosoff M, Kreibich R, Weisz E,
Artwohl M, Kleine OC, Muller M, Baumgartner-Parzer S, Ghysdael J,Freissmuth M, Sexl V.
2004. TYK2 is a key regulator of the surveillance of B lymphoid tumors. J Clin Invest,
114(11):1650-8
Strobl B, Stoiber D, Sexl V, Mueller M. 2011. Tyrosine kinase 2 (TYK2) in cytokine
signalling and host immunity. Front Biosci (Landmark Ed 1), 16:3214-32
Terpe K. 2003. Overview of tag protein fusions: from molecular and biochemical
fundamentals to commercial systems. Appl Microbiol Biotechnol, 60(5):523-33
Tokumasa N1, Suto A, Kagami S, Furuta S, Hirose K, Watanabe N, Saito Y, Shimoda K,
Iwamoto I, Nakajima H. 2007. Expression of Tyk2 in dendritic cells is required for IL-12, IL-
47
23, and IFN-gamma production and the induction of Th1 cell differentiation. Blood,
110(2):553-60
Velazquez L, Fellous M, Stark GR, Pellegrini S. 1992. A protein tyrosine kinase in the
interferon alpha/beta signaling pathway. Cell, 70(2):313-22
Velusamy T, Kiel MJ, Sahasrabuddhe AA, Rolland D, Dixon CA, Bailey NG, Betz BL,
Brown NA, Hristov AC, Wilcox RA, Miranda RN, Medeiros LJ, Jeon YK, Inamadar KV,
Lim MS, Elenitoba-Jhnson KS. 2014. A novel recurrent NPM1-TYK2 gene fusion in
cutaneous CD30-positive lymphoproliferative disorders. Blood, 124(25):3768-71
Venugopal SK, Devaraj S, Jialal I. 2004. RRR-alpha-tocopherol decreases the expression of
the major scavenger receptor, CD36, in human macrophages via inhibition of tyrosine kinase
(Tyk2). Atherosclerosis, 175(2):213-20
Vielnascher RM, Hainzl E, Leitner NR, Rammerstorfer M, Popp D, Witalisz A, Rom R,
Karaghiosoff M, Kolbe T, Müller S, Rülicke T, Lassnig C, Strobl B, Müller M. 2014.
Conditional ablation of TYK2 in immunity to viral infection and tumor surveillance.
Transgenic Res, 23(3):519-29
Waldrop, G. L. 2015. Biotin. eLS. Published Online: 27 JAN 2015
DOI: 10.1002/9780470015902.a0000644.pub2
Wallace C, Smyth DJ, Maisuria-Armer M, Walker NM, Todd JA, Clayton DG. 2010. The
imprinted DLK1-MEG3 gene region on chromosome 14q32.2 alters susceptibility to type 1
diabetes. Nat Genet, 42(1):68-71
Wan J, Fu AK, Ip FC, Ng HK, Hugon J, Page G, Wang JH, Lai KO, Wu Z, Ip NY. 2010.
Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in
Alzheimer's disease. J Neurosci, 30(20):6873-81
Wang CY, Hitz S, Andrade JD, Stewart RJ. 1997. Specific immobilization of firefly
luciferase through a biotin carboxyl carrier protein domain. Anal Biochem, 246(1):133-9
48
Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L, O'Shea JJ. 2004. Signaling by IL-12
and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev, 202:139-56
Waugh DS. 2005. Making the most of affinity tags. Trends Biotechnol, 23(6):316-20
Wolk K, Witte E, Warszawaska K, Sabat R. 2011. Biology of interleukin-22. Semin
Immunopathol, 32(1):17-31
Wood DW. 2014. New trends and affinity tag designs for recombinant protein purification.
Curr Opin Struct Biol, 26:54-61
Yeh TC, Pellegrini S. 1999. The Janus kinase family of protein tyrosine kinases and their role
in signaling. Cell Mol Life Sci, 55(12):1523-34
Zeng B, Li H, Liu Y, Zhang Z, Zhang Y, Yang R. 2008 Tumor-induced suppressor of
cytokine signaling 3 inhibits toll-like receptor 3 signaling in dendritic cells via binding to
tyrosine kinase 2. Cancer Res, 68(13):5397-404
Zheng CF, Simcox T, Xu L, Vaillancourt P. 1997. A new expression vector for high level
protein production, one step purification and direct isotopic labeling of calmodulin-binding
peptide fusion proteins. Gene, 186(1):55-60
Zhu X, Lv J, Yu L, Zhu X, Wu J, Zou S, Jiang S. 2008. Proteomic identification of
differentially-expressed proteins in squamous cervical cancer. Gynecol Oncol, 112(1):248-56
49
7. Appendix
7.1. Vector map of pTyk2/AviTag-puro
Selected features Beta-actin promoter 1 – 1719 (GenBank Access. No. E02194.1)
Tyk2 cDNA 1734 – 5467 (GenBank Access. No. AF173032)
Avitag sequence 5467 – 5517 (www.avidity.com)
Human beta globin 5518 – 6711 (GenBank Access.No. U01317.1)
Puromycin resistance 6712 – 8210 (pKO SelectPuro V810; Stratagene)
Neomycin resistance 8213 – 10239 (pKO SelectNeo; Stratagene)
LoxP site 8218 – 8251; 10198 – 10231 (pEasyFlox; Addgene # 11725)
Ampicillin resistance 11839 – 12498 (pBluescript II KS(-); Stratagene)
All features were either cloned into the backbone vector pBluescript II KS(-) (Stratagene) (www.addgene.org/vector-database/1945/) or were intrinsic features of it.
Vector map generated by ’A plasmid Editor (ApE)’ software (http://biologylabs.utah.edu/jorgensen/wayned/ape)
AviTag 5467..5517human beta globin 5518..6711
LoxP 10198..10231
Neo_R 8213..10239
LoxP 8218..8251Puro_R 6712..8210
Amp_R 11839..12498
pTyk2Avitag
13734 bp
b-actin promoter 1..1719
Tyk2 cDNA 1734..5467
50
7.2 Vector map of pBirA/FLAG-blast
Selected features
Blasticidin resistance 314 – 801 (pLenti6.3/V5-DEST; Invitrogen)
Ampicillin resistance 813 – 1472 (pBluescript II KS(-); Stratagene)
Beta-actin promoter 2709 – 4427 (GenBank Access. No. E02194.1)
BirA cDNA 4442 – 5404 (GenBank Gene ID: 948469)
FLAG-tag 5405 – 5431 (pFLAG-CMV-2; Sigma)
All features were either cloned into the backbone vector pBluescript II KS(-) (Stratagene) (www.addgene.org/vector-database/1945/) or were intrinsic features of it.
Vector map generated by ’A plasmid Editor (ApE)’ software (http://biologylabs.utah.edu/jorgensen/wayned/ape)
Amp_R 813..1472
FLAG 5405..5431
BirA cDNA 4442..5404
pBirAFLAG
7851 bp
Blast_R 314..801
human beta globin 5434..7183
b-actin promoter 2709..4427