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

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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

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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

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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

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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

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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).

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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).

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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).

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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

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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

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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).

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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

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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)

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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).

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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).

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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).

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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

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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)

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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).

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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)

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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.

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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

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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