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Selection and Production of Recombinant Binders for Use in Protein Microarrays

Diploma Thesis 2009 103 Pages

Biology - Micro- and Molecular Biology

Excerpt

TABLE OF CONTENTS

I ABSTRACT

II ABBREVIATIONS

1. INTRODUCTION
1.1 Recombinant binders for proteomics and diagnostics
1.2 Protein microarrays as an application of recombinant binders
1.3 Structure and properties of antibodies and recombinant binders
1.3.1 Antibodies
1.3.2 Single-chain fragment variable (scFv)
1.3.3 Other recombinant binders
1.4 Phage biology
1.4.1 Structure of the Ff phage particle
1.4.2 Structure of the Ff phage genome
1.4.3 The phage lifecycle
1.5 Phage display
1.6 Phage RDA
1.7 Aim of this work

2. MATERIALS AND METHODS
2.1 Materials
2.1.1 Chemicals
2.1.2 Equipment
2.1.3 Consumables
2.1.4 Biochemicals
2.1.5 Kits
2.1.6 Oligonucleotides
2.1.7 Bacteria strains
2.1.8 Phagemid library
2.1.9 Helper phage VCSM13
2.1.10 Plasmid vectors
2.1.11 Buffers, media and solutions
2.2 Methods
2.2.1 Phage display methods
2.2.1.1 Precautions and sterilisation
2.2.1.2 Production of the initial phage library
2.2.1.3 Preparation of phages
2.2.1.4 Panning against immobilized antigens
2.2.1.5 Reamplification of the phage library
2.2.2 General DNA methods
2.2.2.1 Plasmid/phagemid purification
2.2.2.2 Determination of DNA concentration
2.2.2.3 Agarose gel electrophoresis
2.2.2.4 Restriction digestions
2.2.2.5 Subcloning of scFv inserts
2.2.2.6 Sequencing and sequence analysis
2.2.2.7 PCR
2.2.2.7.1 Prevention of contamination and quality control
2.2.2.7.2 Amplification of scFv inserts from plasmids/phagemids
2.2.3 Protein methods
2.2.3.1 SDS-PAGE and staining of gels
2.2.3.2 Western blot
2.2.3.3 Indirect ELISA
2.2.3.4 Purification of scFvs
2.2.3.5 Concentration of growth medium
2.2.3.6 Preparation of bacteria lysates
2.2.3.7 Immobilisation of scFvs on glass slides
2.2.3.7.1 Labelling of antigen
2.2.3.7.2 Immobilisation and on-chip purification
2.2.3.7.3 Blocking, incubation, detection and data analysis
2.2.4 Bacteria methods
2.2.4.1 Preparation of glycerol stocks
2.2.4.2 Expression of scFvs in XL1-Blue
2.2.4.3 Expression of scFvs in AVB100
2.2.4.4 Periplasmic extraction
2.2.4.5 Preparation of competent cells and heat shock transformation
2.2.5 Phage RDA methods
2.2.5.1 Panning against immobilised antigens and background
2.2.5.2 Preparation of phage ssDNA
2.2.5.3 Production of tester and driver
2.2.5.3.1 Amplification of scFv insert
2.2.5.3.2 PCR purification
2.2.5.3.3 XbaI restriction digestion of tester DNA
2.2.5.3.4 Gel extraction of digested tester DNA
2.2.5.3.5 Ligation of adapters to tester DNA
2.2.5.3.6 Production of driver DNA
2.2.5.4 Subtractive hybridisation and processing of samples
2.2.5.4.1 Determination of tester - driver ratio
2.2.5.4.2 Hybridisation of tester and driver
2.2.5.4.3 Fill in reaction
2.2.5.4.4 Digestion of ssDNA
2.2.5.4.5 Amplification of difference product

3. RESULTS
3.1 Phage display
3.1.1 Quality control
3.1.1.1 Transformation efficiency in production of initial phage library
3.1.1.2 Assessment of scFv insert stability
3.1.1.3 BstOI restriction digestion of pooled scFv clones
3.1.1.4 Test for enrichment of phages on antigen coated wells
3.1.2 Screening and first characterisation of selected binders
3.1.2.1 ELISA screen for selected scFvs
3.1.2.2 BstOI restriction digestion of single scFv clones
3.1.2.3 Sequencing and subtype determination of different scFvs
3.2 Expression and characterisation of different scFvs
3.2.1 Different detection methods for scFvs
3.2.1.1 Anti-c-myc-HRP
3.2.1.2 Protein L-HRP
3.2.1.3 Streptavidin-HRP
3.2.1.4 Comparison of the detection methods
3.2.2 Optimisation of expression and scFv yield
3.2.2.1 Periplasmic extract as source of scFvs
3.2.2.2 Effect of sucrose and glycerol on scFv yield
3.2.3 Further characterisation of scFvs
3.2.3.1 Analysis of scFvs from different sources by PAGE and Western blot
3.2.3.2 Analysis of scFv specificity in Western Blot
3.2.3.3 Test for cross reactivity of different scFvs in ELISA
3.3 Purification of scFvs on protein L agarose resin
3.4 Immobilisation of scFvs on glass slides
3.4.1 Control of surface binding properties
3.4.2 On chip purification of scFvs for capture of labelled antigen
3.4.2.1 Detection of labelled gamma globulins by on chip purified scFvs (1)
3.4.2.2 Validation of on-chip purification (1) by ELISA
3.4.2.3 Detection of labelled gamma globulins by on-chip purified scFvs (2)
3.4.2.4 Validation of on-chip purification (2) by ELISA
3.4.2.5 Detection of BSA-FITC by on chip purified scFvs
3.5 Phage-RDA
3.5.1 One round of phage RDA
3.5.2 Optimisation of the initial scFv insert amplification

4. DISCUSSION
4.1 Selection of scFvs by phage display and quality control assays
4.1.1 Transformation efficiency
4.1.2 Instability of the scFv insert
4.1.3 Assays to test for phage enrichment
4.2 Detection methods and affinity tags
4.3 Different sources of scFvs in expression
4.4 Effects of different additives on scFv yield
4.5 Specificity and cross reactivity of scFvs
4.6 Immobilisation of scFvs on glass slides
4.7 Phage RDA
4.8 Outlook
4.8.1 scFv microarrays
4.8.2 Phage RDA

5. REFERENCES

6. APPENDIX

I ABSTRACT

Antibody microarrays hold great promise for the exploration of the human proteome. But one drawback of this method is the limited availability of appropriate affinity reagents which determine the validity of such experiments. Large numbers of antibodies are already available, these however only cover a small part of the whole proteome, and the generation of antibodies for proteome-wide studies with conventional methods would be uneconomic and not sustainable.

Recombinant binders may fill this gap of missing affinity reagents. Their main advantages are that they can be selected in large numbers from antibody libraries and that they do not depend on animal immunisation. These libraries even can be created fully synthetically and allow the properties of the binders herein to be exactly determined.

In this work, the phage display system was applied for selecting recombinant scFv antibodies from such an antibody library. Several binders against gamma globulins and BSA-FITC were selected. The binders were then expressed in different bacteria strains and under different conditions. Different fractions from expression such as growth medium and periplasmic extract were also tested in regard to their scFv content. In addition a number of detection methods were evaluated in regard to specificity and sensitivity in the detection of scFvs. scFvs could also be purified from the expression samples. As the long term aim is to set up a microarray based on recombinant binders, different immobilisation strategies were evaluated including a one step on-chip purification and immobilisation of scFvs from crude expression samples. The on-chip purification was shown to work, but the best results regarding signal intensity were obtained when purified scFvs were immobilised in a directed manner on streptavidin-coated glass slides, as well as in undirected immobilisation of purified scFvs on epoxy-silane slides.

In a side project, the novel phage RDA technique for selection of binders by a PCR and DNA hybridisation based method was applied. Here, mainly the critical steps in the amplification process were addressed.

III ABBREVIATIONS

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1. INTRODUCTION

After finishing the human genome sequence in 2003,63 the functions of most of the genes still remained unknown. Functional analysis of the discovered genes was the next step to be taken on the way to the understanding of the molecular mechanisms of human biology.35 DNA microarrays have until now contributed a great part to the understanding of cellular interaction processes by transcription profiling but also other applications such as epigenetic studies.52 With microarray approaches RNA expression data is still being created on a large scale day by day, and in recent times next generation sequencing technology has sped up this trend.105 As even faster sequencing methods will soon be available, scientists will be able to easily explore whole genomes and transcriptomes.22 Yet high quality quantitative protein expression data, which is essential for understanding cellular processes, is compared to nucleic acid data still more than rare. This is to a great part due to restrictions of the current proteomic tools which don’t allow the exploration of the whole proteome, which has been estimated to comprise more than one million proteins of distinct structural and functional properties.49,77

Liquid chromatography and 2-dimensional gel electrophoresis systems coupled with mass spectrometry are widely used in proteomic research. However these tools are not suited for high throughput testing of complex samples. Antibody microarrays, which are miniaturised systems based on affinity capture of specific analytes, in turn deliver the sensitivity needed for proteomic research and the degree of automation needed for high-throughput screenings. But they are dependent on large numbers of high quality affinity reagents, such as antibodies. Whereas large numbers of protein affinity reagents are available commercially, their specificity and sensitivity often is uncertain.118 In addition, currently available protein affinity reagents cover only a fraction of the human proteome, and their costs are still too high for proteome wide applications.118 In recent times, projects have been initiated to overcome these limitations.18,42,45,118,120 The common goal of these projects is the generation and characterisation of high quality protein affinity reagents against each protein in the human proteome.

1.1 Recombinant binders for proteomics and diagnostics

Since the discoveries of Behring and Kitasato over 100 years ago, it was known that specifically binding molecules could be obtained from blood sera.10 By so-called immunisation of animals with an antigen (molecule which is bound by an antibody), the immune system becomes stimulated and so-called polyclonal antibodies are produced. These are a group of different immunoglobulins that bind with high specificity and affinity to the antigen with which the animal was immunized.64 Polyclonal antibodies have been used for clinical purposes in the past mainly in passive immunisation but also find their use in diagnostics and modern biological research until today.120 Monoclonal antibodies are derived from single immortalised B-cell clones and are thereby directed towards one specific site on an antigen (epitope) only. They were first generated by Köhler et al. in 1975 using the so- called hybridoma technology.69 These antibodies are commonly used in clinical applications but even more extensively in biological research. Production of monoclonal antibodies by hybridoma technology is time consuming and too expensive to be able to supply the numbers and amounts of affinity reagents which are needed for proteome wide studies. In order to explore the full complexity and functions of the human proteome well characterised affinity reagents, also called binders, must be provided on scales which exceed the conventional methods for affinity reagent generation. The large numbers of binders which already exist cover only a small fraction of the proteome, and this often with high redundancies (for example, >900 antibodies against p53) whereas no binders are available for the vast majority of the proteome.118 Further these binders are often not sufficiently characterised regarding their specificity, affinity and usability in different applications.

Recombinant binders may meet the demands for proteome wide coverage of affinity reagents, as they are independent of animal immunisation and labour intensive hybridoma technology. In the recombinant binder approach, antibody genes are created synthetically or cloned from animals or humans into large libraries from which the binders then can be selected. These selection processes are in contrast to hybridoma technology highly automatable70,104,119,121 and the expression of the selected binders was optimised for E. coli or yeast. A large scale antibody generation initiative for example is the antibody factory (antibody-factory.de) which aims at delivering antibodies on demand to the scientific community.

1.2 Protein microarrays as an application of recombinant binders

Antibody microarrays are analytical systems in which the capture reagents, mostly antibodies, are applied onto a solid support in a miniaturized format in oder to capture specific antigens.74,124,126 Although antibody microarrays were introduced after DNA microarrays, the feasibility of miniaturized and multiplexed immunoassays was already reported in the late 1980s.36,37 Signals are derived from the captured antigens mainly by fluorescence detection, whereas the signal intensity represents the amount of the captured analyte. Sensitivities in the picomole to femtomole range have been reported.74,123

Although still under development, the antibody microarray technology has already shown wide application potential for clinical cancer research and diagnostics.2,102 The number of binders in such oncoproteomic profilings vary from a few tens,1,72 to hundreds, as in studies focused on a more global protein expression analysis.30,48,93

With the increasing number of analytes in these diagnostic or proteomic microarray studies the availability of high quality binders is becoming the limiting factor. Proteome wide coverage on an antibody array is only feasible by using recombinant antibodies, which have already been shown to be able to fulfil their function in the antibody microarray format.127

1.3 Structure and properties of antibodies and recombinant binders

1.3.1 Antibodies

Antibodies, also called immunoglobulins (Ig), are present in humans in five forms which mainly differ in the constant region of the heavy chain. These subforms are called IgM, IgD, IgG, IgA and IgE whereas IgGs are further subdivided into IgG1, IgG2, IgG3 and IgG4.64 Different forms of immunoglobulins appear in other animals, for example so-called IgY in chickens,80 IgW in some sharks and lung fish11 and the camelid antibodies which posses a completely different structure.91 A typical IgG antibody is built up out of four protein chains, two identical heavy chains (~50kDa), and two identical light chains (~25kDa) (Fig. 1.1). These protein chains interact by their interface regions and are further stabilised by disulfide bonds to form a Y-shaped molecule. The two identical antigen binding sites are found at the upper tips of the arms. They enable the antibody to simultaneously bind to two identical structures. Two types of light chains, the lambda and kappa chains, are found in antibodies, whereas one antibody only has either only kappa or lambda light chains. No functional difference has been found between antibodies having a lambda or kappa light chain.64 IgG antibodies exhibit high stability, high specificities and affinities as they undergo the so-called affinity maturation during generation in the body.64 The Fc (fragment crystallisable) part of the antibody (Fig. 1.1 a) differs between the antibody subtypes. In the body it enables recognition and uptake of for example pathogens by cells carrying specific receptors, such as macrophages.

Digestion of antibodies with the protease papain results in two so-called Fab fragments (Fig. 1.1 a) and the Fc fragment. The Fab fragment, and its derivates such as the scFv (singlechain fragment variable) however also can be generated recombinantly.97

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Fig. 1.1: Schematic representations of an IgG antibody. a) Representation showing the basic structure and the functional parts of an IgG antibody. Fab: Fragment antigen binding, Fc: Fragment crystallisable, Fv: Fragment variable. b) Representation showing the conformation of the antibody Ig domains in the antibody. VH and VL: Variable regions of the heavy and light chains respectively, CH and CL: Constant domains of the heavy and light chains respectively. Pictures adapted from Janeway et al.64

1.3.2 Single-chain fragment variable (scFv)

The single-chain fragment variable (scFv) is a recombinant fusion protein consisting of the heavy and light chain variable region of an antibody fused together by a glycine-serine linker peptide of about 15-25 amino acids.41,61,62 This molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. By linking the two variable fragments together, expression in E. coli could be improved in comparison to the Fv fragment.14,61 scFvs show lower affinities than the corresponding Fab fragments.15 However, since the Fab fragment is double the molecular size, and requires the production and connection of two different polypeptides via a disulfide bond, folding and assembly of Fab fragments in the periplasm of E. coli is less efficient than for scFvs.107 Therefore the scFv antibody fragments are widely used today, mainly in phage display (section 1.5). Another advantage of scFvs is their small size which enables better tissue penetration in clinical use, and their longer retention times in the body.34

1.3.3 Other recombinant binders

In the past, affinity reagents were almost uniquely based on antibody scaffolds such as the scFv, Fab and variations of these.59 These recombinant antibodies are in part also found in clinical applications and are the fastest growing group of pharmaceuticals.34 In more recent times a novel group of affinity reagents which are independent of antibody fragments have evolved. These affinity reagents are mostly based on fully synthetic protein scaffolds. Examples of these new affinity reagents are peptide aptamers,27 affibodies,94 armadillo repeat proteins,95 or darpins.117 These completely recombinant non-immunoglobulin affinity reagents have several advantages over the antibody based ones such as their small size, high affinities and stabilities. Thereby they may be better suited for clinical applications.13,56,108

1.4 Phage biology

The filamentous bacteriophages (genus Inovirus), are a group of viruses which contain a circular single-stranded DNA (ssDNA) positive strand genome encapsulated by a long protein cylinder. The Ff class of the filamentous phages, the f1, fd and M13 phage, have been studied in detail.84,88 After infection, phages use their host bacteria to replicate but do not kill them during that process. The bacteria tolerate the infection and continue growing at about half the rate as prior to infection. About 200 phage particles are produced per cell and generation, resulting in high phage titers.

Because of their replication mode and structure, phages are a valuable tool for biological research for example as cloning vehicles, for studying recombination and DNA repair but most prominently for selection of binders and ligands by phage display.65

1.4.1 Structure of the Ff phage particle

The Ff phage particle is about 6.5nm in diameter and 939nm in length.8,84 The mass of the particle is about 16.3MDa of which 87% is contributed by protein. The circular single- stranded DNA genome is encased in a somewhat flexible protein cylinder which consists of about ~2700 molecules of the 50 amino acid major coat protein pVIII. The 10-13 carboxy terminal residues of pVIII form the inside of the wall of the cylinder and interact with the sugar phosphate backbone of the DNA. On the one end of the phage there are about five molecules, each of pVII and pIX, which bind to the packaging signal of the genome. The other end is made up of about five molecules each of pIII and pVI. pIII, which carries the recombinant protein in most phage display systems, is made up of three domains, N1, N2 and CT, separated by glycine-rich regions. The amino terminal domain N1 is required during infection for the translocation of the DNA into the cytoplasm and the insertion of the coat proteins into the membrane. Domain N2 is responsible for binding of the bacterial F-pilus. Removal of domains N1 and N2, by protease treatment, results in non-infectious phage particles. The third domain (CT) is essential for forming a stable phage particle.

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Fig. 1.2: Structure of the Ff bacteriophage particle.

a) Electron micrograph of a filamentous bacteriophage b) Schematic representation of the structure of filamentous bacteriophage describing its coat morphology with the five coat proteins, pIII, pVI, pVIII, pVII and pIX and the circular single-stranded DNA geome. Pictures from Stolle, T.O.130

1.4.2 Structure of the Ff phage genome

The genomes of the Ff phages M13, f1 and fd have been sequenced completely. Their DNA sequences are 98% homologous, and thus the protein sequences of the gene products are practically the same. The genomes comprise ~6400 nucleotides encoding 11 genes (Tab. 1.1).8,88 The genes are grouped in the genome according to their function in the life cycle of the phage (Fig.1.3). The one group consisting of genes II, V and X, is required for the replication of the phage genome. A second group (genes VII, IX, VIII, III and VI) code for the capsid proteins. The third group of genes encodes the proteins, that are involved in the membrane-associated assembly of the phage particle (genes I, XI and IV).

The intergenic region in the phage genome does not code for protein. It contains the packaging signal and the origins for the synthesis of the viral (+) and complementary (-) DNA. Transcription uses the (-) strand of the double-stranded replicative intermediate form. In viral DNA replication, thus the original (+) strand which enters the bacteria cell contains the coding sequence. The two strong terminators divide the genome into two transcription regions: a frequently transcribed region containing genes III through VIII, and an infrequently transcribed region containing genes III through IV.

Tab. 1.1: Genes and gene products of the f1 bacteriophage (adapted from Barbas 3rd et al.8 )

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Fig. 1.3: Genome of the Ff bacteriophage. Relative positions of the genes (gene I - gene XI) and important promoters and terminators are shown. IR: Intergenic region, T: Two strong terminators, t: Weak terminator in gene I, GA, GB and GH: Promoters of the frequently transcribed region, P: Promoter of the infrequently transcribed region, Picture from Stolle, T.O.130

1.4.3 The phage lifecycle

The lifecycle of the bacteriophage can be divided into three steps: Infection, replication of genome, and assembly and export. The infection of bacteria with Ff phages requires the interaction of the phage surface protein pIII with the F pilus of the bacteria. The proteins required for the F pilus structure, assembly and disassembly are encoded by genes on the F conjugative plasmid.38

Besides the F pilus a number of other proteins are required for successful infection with the phage. These proteins, TolQ, TolR, and TolA, are located in the cytoplasmic membrane and are necessary for maintaining the integrity of the bacterial outer membrane.78 Infection is initiated by binding of the N2 domain of pIII to the tip of the F pilus.116 After binding of N2 to the pilus, the pilus retracts thus bringing the pIII to the periplasm and allowing the N1 domain to interact with TolA which acts as a co receptor101. After further protein interaction steps, the phage particle is then able to fuse with the membrane and inject its ssDNA genome into the host cell.

Once the viral (+) strand DNA enters the cytoplasm, the complementary (-) strand is synthesized by bacterial enzymes (Fig 1.4). This double stranded DNA is called the parental replicative form (RF) DNA. The (-) strand of the RF is the template for transcription. Replication of the RF functions through pII, which nicks the (+) strand of the RF at a specific place in the intergenic region. The resulting 3’-hydroxyl acts as a primer for synthesis of a new viral strand via rolling-circle replication using bacterial enzymes. After one round, pII circularizes the displaced (+) strand DNA, which then again is turned into an RF molecule by bacterial enzymes. This way a pool of double stranded RF molecules is generated. RF DNA synthesis continues until the amount of pV, which is synthesised in parallel, reaches a critical concentration and the pV molecules binds to the viral (+) ssDNA, thus inhibiting its conversion into RF DNA. The 78-nucleotide hairpin packaging signal is at one end of the pV- ssDNA complex.

All phage proteins except pII, pX and pV, which reside in the cytoplasm, are inserted into the cytoplasmic- and outer membrane, either by spontaneous insertion or by active transport directed by their signal peptides. The packaging signal of the pV-ssDNA complex then interacts with these proteins, and finally the ssDNA is extruded from the cytoplasm into the extracellular space while pV simultaneously disassociates from the ssDNA and the phage genome is encapsulated by many copies of pVIII. Thereby new phage particles get generated without irreversibly damaging the host cell.

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Fig. 1.4: The phage lifecycle. a) E. coli host cell is infected by the filamentous phage via the F-pilus.

b) After insertion of the ssDNA genome into the cytoplasm the ssDNA molecule is converted into the double stranded replicative form (RF) and multiplied. Replication generates about 100 double- stranded copies. c) New ssDNA is synthesised from the RF DNA. d) RF DNA also serves as a template for transcription of the phage genes. Phage proteins facilitating replication reside in the cytoplasm whereas proteins for packaging are inserted into the inner and outer plasma membrane. e) ssDNA interacts via the packaging signals with the phage proteins in the plasma membrane and is extruded into the extracellular space in a non-lytic manner while being simultaneously encapsulated by envelope proteins. Picture from “QIAprep M13 Handbook” (Qiagen, 2002)

1.5 Phage display

Numerous methodologies are available for selecting recombinant antibodies from libraries such as ribosome display,47 bacterial surface display,112 and yeast surface display.21 Unlike classical immunisation and hybridoma technology these methods allow isolation of antibodies against almost all types of antigens, even against molecules with strong toxic effects on animals which could not be used in animal immunisation.

One of the most powerful tools and the most widely used library methodology however is phage display (Fig. 1.5).9,54,65,86,109,110 In this method DNA fragments encoding large libraries of peptides, ORFs or affinity reagents, are cloned into phage vectors allowing display on the surface of phage particles. The ligands are usually displayed as a fusion protein with the surface proteins pIII or pVIII. Their sequence information is contained in the same phage particle displaying the ligand. Selection of specific ligands is then achieved by a so-called panning step. In this step the phages displaying the library are typically incubated on microtiter wells which are coated with the antigen of interest. Phage particles which carry a ligand binding to the antigen are captured on the surface, and phages which don’t bind or don’t bind strongly enough are removed in the subsequent stringent washing steps. Phages which are bound to the antigen are then eluted, for example under acidic conditions, and used to infect E. coli bacteria hosts and re-grow a new library of phages. This library with reduced complexity contains the binders that bound to the antigen in the first panning step but, also many phages which bound unspecifically. Therefore the panning with the library of reduced complexity is usually repeated three to four times usually resulting in a strong enrichment of phages carrying a specific binder for the antigen against which it was selected. Initial phage vectors carried all the genetic information required for the phage life cycle,12,25,86,109,129 but nowadays phagemid vectors have become the more popular vector system for phage display.9,23,55 Phagemids are small plasmid vectors which can carry large inserts and can be transformed into E. coli with high efficiencies. Therefore they are ideally suited for the generation of large peptide or protein libraries. These phagemid vectors contain the filamentous phage intergenic region with the origin of replication for viral and complementary strand synthesis as well as the hairpin packaging signal. The phagemid can maintain itself as a plasmid, and also enables inducible expression of the ligand originally displayed as fusion protein. As the phagemid vector contains no other phage proteins than the pIII fusion protein, particles carrying phagemids cannot replicate autonomously which prevents uncontrolled spreading of phages in the laboratory. Therefore a so-called helper phage is needed for generation of the phage particles. The helper phage provides all other functions of filamentous phages and also possesses the wildtype pIII protein. However, since the helper phage genome has a reduced packaging rate the phagemid is preferably packaged into the generated particles. Resulting phages do not only carry the phagemid ssDNA and the wildtype pIII protein but also the pIII-fusion protein on their surface (~10 % of the phages).71,82

After enrichment of specific ligands these can be expressed from the phagemid by induction of a separate promoter. The key to expression in E. coli is the direction of the antibody fragments to the periplasmic space, where the oxidizing environment allows the formation of disulfide bonds.109

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Fig. 1.5: Principle of phage display. a) Panning of the initial phage library. Phages carrying binders on their surface are incubated on antigen coated wells. b) Elution of bound phages from the surface of the microtiter well. c) Infection of E. coli cells with the eluted phages carrying the phagemids encoding for the ligand on the same particle. d) Superinfection of E. coli cells with helper phages results in production of a new library with reduced complexity. e) Preparation of phages from the bacteria culture. f) Panning of the new phage library on antigen coated wells (same antigen as in a). Steps b) to

f) are repeated for a total of three to five panning rounds (including initial panning). g) Bacteria clones infected with the phage output are selected and screened for expression of binders against the antigen on which they were selected. Positive clones can be used for production of monoclonal binders.

1.6 Phage RDA

Subtractive approaches to phage display which aim at generating binders against differentially represented antigens in two mixtures have been established.43,51,113,114 But as they are all based on phage display they lack sensitivity mainly due to a bias in the library reamplification steps in which some antibody fragments are expressed more effectively than others. Ineffective enrichment of high affinity binders due to inefficient elution from the wells also is an additional problem inherent to phage display. By this, binders with the highest affinity may be lost. Phage RDA is a novel application of representational difference analysis (RDA)81 and aims at circumventing these problems.

Representational difference analysis (RDA), a method for detecting differences between two DNA populations, has been applied in comparing cDNA populations from cancer and healthy tissue44 or for comparison of microbial genomes.81 The method has also been improved, mostly in regard to specificity and sensitivity.16

In phage RDA, the subtractive approach of RDA is used for displaying the differences in the antibody repertoire of two phage populations which were incubated on different antigens. The vision for phage RDA is the detection of differentially expressed or modified proteins in the comparison of healthy and diseased tissue. With this approach novel biomarkers that cannot be detected on nucleic acid level could be found. A successful phage RDA selection would result in binders that recognise the differentially represented antigens in the two samples compared and could then be used for the identification of these factors and for further studies.

The process of phage RDA (Fig. 1.6) starts with the panning of a phage library on two microtiter wells coated with the samples to be compared. Elution of the complete phage DNA from the wells by lysis of the phage particles circumvents the elution problems inherent to phage display, where binders with very high affinities cannot be eluted effectively without damaging the phage particle and thus preventing successful re-infection. The binder DNA inserts are then amplified by PCR resulting in two DNA populations, the so-called tester and driver DNA. DNA adapters are then ligated to the tester DNA but not to the driver DNA. In a subtractive hybridisation of tester and driver, to which the driver is given in excess, DNA fragments which are present in both DNA populations and therefore represent non- differentially represented binders, form tester driver hybrids. Binder inserts which are present only in one of the DNA populations can only hybridise within their own group. This hybridisation thus results in three classes of DNA fragments: Tester-driver hybrids, tester- tester hybrids or driver-driver hybrids. Due to the fact that only the tester carries the DNA adapters, tester-tester hybrids are exponentially amplified in a subsequent PCR reaction with adapter specific primers, whereas tester-driver hybrids only are amplified in a linear manner, and driver-driver hybrids are not amplified at all. The product of the PCR amplification then is used in a second and third round of subtractive hybridisation, with a new set of adapters in each round. After three rounds of RDA the resulting binder fragments can be cloned and analysed by restriction digestion. In a setting in which phage RDA would be used to discover new biomarkers, Western blot analysis and co-immunoprecipitation using the selected binders as bait could be used for identifying the targets of the isolated binders.

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Fig. 1.6: Principle of phage RDA. a) Phage ssDNA is eluted from the wells after panning of a high complexity phage library against immobilised antigens. b) Binder inserts are amplified by PCR from the eluted phage DNA. c) The tester DNA is modified with an adapter whereas the driver DNA is not modified. d) Tester and driver DNA are hybridised in a reaction in which driver DNA is in excess. e) Fill in reaction with hybridisation products and digestion of single-stranded DNA by mung bean nuclease. f) PCR amplification of hybridisation products with adapter specific primers. Tester-tester hybrids are exponentially amplified whereas tester-driver hybrids are only amplified linearly and driver-driver hybrids are not amplified at all due to lack of adapters. The product from f) is then modified with a new set of adapters and now represents the tester sample for the next subtractive hybridisation with the driver DNA. g) After a total of three rounds the product from f) is cloned into a vector and selected binders can be screened.

1.7 Aim of this work

With increasing need of larger numbers of high quality binders for microarray applications, selection and characterisation of such binders from recombinant antibody libraries is a possible means of satisfying this need. Phage display is a method which allows such binder selection and enables subsequent expression and characterisation of the selected binders. The aim of this work was to establish and evaluate methods for the selection, expression and purification of recombinant scFv binders. Selected binders were then to be functionally immobilised onto a solid support for capture of antigen. Therefore different binding strategies were to be evaluated.

A side project in this thesis was the application and further development of the phage RDA method, which still is in its infancy. The method should be applied in selection of binders against a single antigen and critical steps in the process should be addressed.

2. MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemicals

Tab. 2.1: Chemicals and ready to use solutions

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2.1.2 Equipment

Tab. 2.2: Technical equipment

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2.1.3 Consumables

Tab. 2.3: Consumables (plasticware, filters, glass slides, disposables)

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2.1.4 Biochemicals

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Tab. 2.4: Biochemicals (enzymes, antigens, affinity reagents, protein and DNA ladders)

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2.1.5 Kits

Tab. 2.5: Kits for DNA and scFv preparation

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2.1.6 Oligonucleotides

Tab. 2.6: Oligonucleotide sequences. All oligonucleotides were purchased from Thermo Scientific and diluted in nuclease free water to a final concentration of 100mM before use

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2.1.7 Bacteria strains

Tab. 2.7: Bacteria strains

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2.1.8 Phagemid library

The naive human scFv antibody phage library used in this work was generated by VTT Biotechnology (Espoo, Finland). It has been used to isolate antibodies against different antigens.98,100,119,122 For generation of the library the antibody genes had been cloned from a pool of human lymphocytes of 50 healthy donors. After mRNA isolation and cDNA synthesis, the repertoire containing the variable regions of heavy chains (VH) was prepared by amplifying the IgM-specific cDNA. The light chain repertoires were obtained by amplifying both kappa (Vκ) and lambda (Vλ) specific cDNAs separately. The heavy chains were combined with the light chains and cloned into a modified version of the vector pBluescript(SK+) (Stratagene) (further referred to as pBluescript) to create two libraries, VH- Vκ and VH-Vλ, both containing approximately 108 different clones. In the following text the libraries are referred to as kappa and lambda library respectively.

2.1.9 Helper phage VCSM13

Helper phage VCSM13 (Stratagene, La Jolla, USA) was prepared previously to this work according to Barbas 3rd et al.8 The solution had a phage titer of ~1013 pfu/mL.

2.1.10 Plasmid vectors

Tab 2.8: Plasmid vectors

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2.1.11 Buffers, media and solutions

All solutions, if not stated otherwise, were prepared with Milli-Q water according to the recipes in the tables 2.9 - 2.17. All media and solutions for bacteria and phage culture were either autoclaved or sterilized by filtration. All solutions were stored at RT if not stated otherwise.

Tab 2.9: Media and solutions for bacteria culture and phage display

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Tab. 2.10: Solutions for DNA electrophoresis

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Tab 2.11: Solutions for SDS PAGE

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Tab. 2.12: Recipe for SDS poly-acrylamide gels. The amount in this recipe is sufficient for two 8cm(W) x 7.3cm(H) x 0.75mm(D) sized gels

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Tab. 2.13: Solutions for silver staining of poly-acrylamide gels

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Tab. 2.14: Solutions for Western Blot

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Tab. 2.15: Solutions for ELISA

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Tab. 2.16: Solutions for immobilisation of scFv on glass slides

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Tab. 2.17: Solutions for phage RDA

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[...]

Details

Pages
103
Year
2009
ISBN (eBook)
9783640994267
ISBN (Book)
9783640995226
File size
20.8 MB
Language
English
Catalog Number
v177653
Institution / College
German Center of Cancer Research Heidelberg
Grade
1,1
Tags
microarray protein phage display protein microarray recombinant binder affinity molecule scFv antibody biotinylation western blot ELISA proteomics FITC Cy3 Cy5 RDA PCR representational difference analysis phage RDA phage cloning protein A on-chip purification protein expression scFv purification molecular biology

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Title: Selection and Production of Recombinant Binders for Use in Protein Microarrays