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ProteomeBinders: Planning a European resource of affinity reagents for analysis of the human proteome

February 2007
Nature Methods 4(1):13-7

DOI:10.1038/nmeth0107-13

Source
PubMed

Authors:

Carl A K Borrebaeck

Lund University

Andrew R M Bradbury

Los Alamos National Laboratory

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ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination. NOTE: In the version of the article originally published, Manfred Koegl’s name was misspelled. Additionally, Zoltan Konthur's affiliation was listed incorrectly; it should be Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany. These errors have been corrected in the HTML and PDF versions of the article.

| Validation criteria and methods for proteome binding molecules (adapted from ref. 7)

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NATURE METHODS

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JANUARY 2007

COMMENTARY

ProteomeBinders: planning a European resource of

affinity reagents for analysis of the human proteome

Michael J Taussig

, Oda Stoevesandt

, Carl A K Borrebaeck

, Andrew R Bradbury

, Dolores Cahill

Christian Cambillau

, Antoine de Daruvar

, Stefan Dübel

, Jutta Eichler

, Ronald Frank

, Toby J Gibson

David Gloriam

, Larry Gold

, Friedrich W Herberg

, Henning Hermjakob

, Jörg D Hoheisel

, Thomas O Joos

Olli Kallioniemi

, Manfred Koegl

, Zoltán Konthur

, Bernhard Korn

, Elisabeth Kremmer

, Sylvia Krobitsch

Ulf Landegren

, Silvère van der Maarel

, John McCafferty

, Serge Muyldermans

, Per-Åke Nygren

Sandrine Palcy

, Andreas Plückthun

, Bojan Polic

, Michael Przybylski

, Petri Saviranta

, Alan Sawyer

David J Sherman

, Arne Skerra

, Markus Templin

, Marius Ueffing

& Mathias Uhlén

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-

characterized affinity reagents, including but not limited to antibodies, for analysis of the human

proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but

nevertheless feasible in the context of a pan-European or even worldwide coordination.

To explore the full complexity and func-

tion of the human proteome, it is essential

to establish a comprehensive, characterized

and standardized collection of specific bind-

ing molecules (‘binders’) directed against

all individual human proteins, including

variant forms and modifications. Primed

with the knowledge of the human genome,

such a systematic bank of affinity reagents

would be a crucial precompetitive resource

to understand and exploit the proteome

Yet although affinity reagents are undeni-

ably of central importance for proteomics,

they presently cover only a very small frac-

tion of the proteome, and even though there

are many antibodies against some targets

(for example, >900 antibodies against p53),

there are none against the vast majority of

proteins. Moreover, widely accepted stan-

dards for binder characterization are vir-

tually nonexistent. Establishing a binder

collection will not be an end in itself, but

must be accompanied by development of

high-throughput assay systems and new-

generation protein-detection technologies.

The benefits would include cost-effective

reagent production and access as well as

improved interlaboratory reproducibility,

and will have an impact on basic research

and medicine as well as the biotechnology

and pharmaceutical industries.

ProteomeBinders: vision and goals

ProteomeBinders is a new European consor-

tium with the vision of establishing an infra-

structure resource of binding molecules for

the entire human proteome, together with

tools for their use and applications in study-

ing proteome function and organization.

When mature, the resource could be simi-

lar in nature to the American Type Culture

Collection (ATCC) for cell lines, making the

reagents available at cost and with no restric-

tions for research use. In this Commentary,

we present the long-term goals of the

ProteomeBinders initiative as well as the

current activities of the consortium. The

current 4-year, 1.8M€ ($2.37M) initia-

tive, funded by the European Commission

Framework Programme in the area of

Research Infrastructures, is a ‘Coordination

Action’ involving a network of 26 EU and 2

US partner institutions, leaders in the area of

affinity-reagent production, characterization

and application (see Supplementary Table 1

online for a list of the lead participants in the

ProteomeBinders consortium).

The consortium will coordinate several

complementary activities:

1. Assessing the resources and methods

required to develop a complete collection

of binding molecules, from representa-

tion of the proteome in cDNA collections

to binder selection and production.

2. Reviewing the properties of different

molecular types of binders, including

natural and recombinant antibodies, scaf-

fold domains, peptides and nucleic acid

aptamers, and linking them with pro-

teomics tools and applications in research,

diagnostics and therapeutics.

3. Establishing criteria and methods for uni-

versally applicable quality assessment and

validation.

4. Defining standards for data representa-

tion and establishing a bioinformatics

platform to display information on char-

acterization of individual binders.

5. Planning the long-term production strat-

egy and organization of the binder infra-

structure.

The consortium will hold regular open

workshops and disseminate information on

the results of its activities through its web-

site (http://www.proteomebinders.org),

which will also contain a list of quality-

assured binding reagents from all sources.

In subsequent phases, the consortium aims

to benchmark different types of binders and

Affiliations are listed at the end of the paper.

e-mail: mike.taussig@bbsrc.ac.uk

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production methods against defined sets of

proteins to select those most appropriate

for various applications. Embarking on the

task of assembling the resource by system-

atically collecting and/or creating the tens

of thousands of reagents needed will require

an application for much more substantial

funds, for example, from the next European

Commission Framework Program, FP7,

starting in 2007.

Scale of the problem

The size of the human proteome (including

splice variants, post-translational modifica-

tions, polymorphisms) is generally recog-

nized to be at least an order of magnitude

greater than the ~24,000 protein-coding

genes (http://www.ensembl.org). This

raises several central questions, presently

being debated within the ProteomeBinders

consortium: ‘Is comprehensive coverage of

the proteome realistic?’ ‘How should tar-

gets be prioritized⎯by biological or medi-

cal relevance, by unknown function, or

according to other criteria (chromosome,

etc)?’ Another factor in the scale of the task

is that several binders against each target

will be required, depending on the nature

of the samples (denatured or native), the

biological status of the protein (post-trans-

lationally modified or not) or the detection

mode of the assay (for example, sandwich

configurations).

Choices and challenges in developing a

binder collection

The consortium will consider the following

stages in binder production and application

to reach a consensus for future action.

Target generat i on. Full-length proteins,

probably the optimal targets for binder

selection, can be expressed from cDNA col-

lections

using a variety of systems (bac-

terial

, insect, mammalian, cell-free), but

there is often limited success in obtaining

them in soluble, correctly folded form

4,5

Protein fragments or peptides are alterna-

tives, especially combined with large-scale

epitope prediction. ‘Protein epitope signa-

ture tags’ (PrESTs)

are genome-unique,

nonrepetitive and nonhydrophobic protein

subsequences, shown in the Swedish human

proteome atlas project to be suitable for

raising and affinity purifying polyclonal

antibodies (http://www.proteinatlas.org)

ProteomeBinders aims to integrate existing,

but presently fragmented, bioinformatics

tools into distributed and/or virtual facili-

ties that specifically address target-site selec-

tion within proteins. In the context of con-

structing a large-scale binder resource, an

important goal will be to reduce the amount

of target required (and hence also the cost),

for example, by using microarrays for selec-

tion or using micro- or nanotechnology for

specificity and affinity assays

Molecular varieties of binders: antibodies

and alternatives. Antibodies are by far the

most familiar and best understood affin-

ity reagents⎯and generally the research-

er’s first choice⎯but not the only ones.

ProteomeBinders unites expertise both on

antibodies and alternative binding reagents

with antibody-like specificity and affinity,

including nonimmunoglobulin scaffolds

9,10

(Affibodies

, Anticalins

, designed Ankyrin

repeat proteins

and others), nucleic acid

aptamers

, peptides and chemical entities

(Box 1). Antibodies (including monoclo-

nals, monospecific polyclonals, camelid

heavy chains

, and recombinant scFv frag-

ments and single domains) are considerably

more difficult to produce compared to high-

yield bacterial expression of some alternative

scaffolds

. The latter are also more robust

and provide opportunities for engineering

of functional properties. Among the attrac-

tions of antibodies are widespread compe-

tence in technologies, access to large libraries

for recombinant selection

and availability

of secondary reagents and detection systems.

Recombinant binding molecules have the

advantage of being completely described

by their sequence, so that documentation

and replication of experiments can be more

objective. Though there may be a problem of

wide acceptance of the alternatives to anti-

bodies, users may well not be too concerned

with the structure of the reagent, so long as

it has been demonstrated to work in their

particular application. Accordingly, the con-

sortium will undertake benchmarking of the

properties of alternative binders alongside

conventional antibodies to define the ‘right

binder for the job’. Whatever the molecular

species, sustainability will be a key factor: ulti-

mately, a replenishable resource is required.

Binder production methods and scaling. The

production of binding molecules for a sys-

tematic program can be contrasted in many

BOX 1 DIFFERENT TYPES OF AFFINITY BINDERS

Antibodies and their fragments are the most familiar and widely used binding

reagents. In comparison, protein scaffolds are often more robust (for example, in

storage, reuse on columns) and give higher production yields in bacterial expression

systems, partly due to the lack of cysteine residues. Aptamers stand out as being

nucleic acid–based binding molecules, permitting simple production by synthesis or

PCR, and distributable as sequence information. Libraries of small-molecule binders

and peptides are accessible through combinatorial synthesis. Compared to a whole

immunoglobulin, alternative binders are small, resulting in less steric hindrance,

and are more readily used in intracellular applications. For the non–immunoglobulin

binder formats, however, expertise is often limited to single laboratories and few

libraries exist for distribution.

Inclusion into the yellow oval in the image indicates compatibility with in vitro

selection methods (see Box 2).

Antibodies

Recombinant

immunoglobulin fragments

Fab

scFv

Alternative

protein scaffolds

DNA or RNA

aptamers

Small molecules

Peptides Organics

Nonimmunoglobulin binders

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respects with hypothesis-driven research

(Table 1). For ‘classical’ antibodies, the pro-

duction routes are either to raise polyclonals

(purified for monospecificity) or hybridomas;

throughput of monoclonals can be increased

by immunization with antigen mixtures and

selections on protein arrays

. For all molec-

ular binder varieties, recombinant display

library approaches can be applied and cou-

pled with several possible selection methods.

Systems with established track records such

as phage display

, ribosome display

19,20

, cell-

surface display, bacterial two-hybrid, func-

tional colony screening, protein-fragment

complementation

, SELEX

for aptamer

selection and combinations of these meth-

ods

will be compared taking into account

the molecular entity being selected and the

intended downstream applications (Box 2).

For example, if affinity is crucial, technologies

with built-in evolution will be required (for

example, ribosome display

), whereas if only

‘some binder at some epitope’ is needed, more

technologies become available and selection

is less stringent. For intracellular applications,

binders should fold functionally in the reduc-

ing environment of the cell

. Other technol-

ogy evaluation criteria include robustness,

library creation and size, range of scaffolds

that can be expressed, automation, and lim-

its of scale and throughput. Contributions

of the consortium will be to deliver effective

protocols and actively check their robustness

by rotating and annotating them between

laboratories, and to identify potentially auto-

matable steps, distinguishing those that are

generic from the method-specific ones.

Characterization and quality control. The

critical area of quality control is all too often

sidelined. Validation will be a central issue;

there will be a requirement to demonstrate

the quality of selection methods and binder

formats, as well as of each individual binder.

Although different binder types may have

superior characteristics for defined applica-

tions, certain criteria (affinity, specificity

and cross-reactivity, native or denatured

target, stability in vivo and in vitro, associa-

tion and dissociation rate constants

26,27

)

are applicable across all formats. The con-

sortium will establish reference criteria

for binder quality control and validation,

which could eventually become a ‘gold stan-

dard’ in the research and commercial areas.

Besides the classical tests of performance,

for example, ELISA and western blotting,

other validation methods range from pro-

tein and tissue microarrays to genomic

correlations with transcript levels, gene

knockouts, transgenesis and bioinformatic

predictions (Table 2)

. High-throughput

systems for initial specificity screens may be

combined with advanced kinetic analyses

for binder optimization

Linking binders to tools and applications.

The area of application is perhaps the most

important criterion in choosing binder

type, selection technology and character-

ization methods. Binder uses include high-

throughput array methods (capture, tissue,

lysate arrays) as well as the more classical

techniques. For diagnostic and prognostic

purposes, the pattern of information

that

can be gained from target-binder interac-

tion is more important than specificity of

the binding event, as long as reproducibil-

ity and correlation with disease are high.

In contrast, for functional analyses, where

the global proteome-wide approach is par-

ticularly applicable, specificity is essential.

Accordingly, the parameters by which bind-

ers have to be evaluated can be very differ-

ent. It is imperative to define applications

beforehand and design or refine the selec-

tion process accordingly.

Novel methods to measure large sets

of proteins using affinity reagents are

becoming available, for example, fluo-

rescence cross-correlation spectrosco-

and proximity ligation approaches,

coupled with DNA amplification

31–33

Further new-generation techniques must

be evolved, particularly to improve sen-

sitivity and specificity of detection (ulti-

mately down to the single-molecule or

single-cell level), the ability to perform

highly multiplexed assays in individual

samples, and to determine the spatial dis-

tribution of large numbers of molecules

in cells and tissues.

Bioinformatics resources. ProteomeBinders

will develop community standards for binder

data representation in collaboration with the

Human Proteome Organization (HUPO)

Proteomics Standards Initiative

(http://

psidev.sourceforge.net/). Crucial param-

eters of binder-target interactions have to be

identified and an ontology of binder proper-

ties formally defined. These standards will be

implemented in a comprehensive database

of binders and other web resources, ideally

in collaboration with other major binder

providers. The database structure needs to

anticipate the information to be captured, to

allow retrieval of binders matching certain

criteria and allow users a meaningful assess-

ment of their performance. Basic search-

able information should include the gene

identifier of the target, the form of the target

used for the binder generation or selection,

Table 1 | Comparison of binder generation for ‘classical’ hypothesis-driven research and systematic approaches (adapted from ref. 36)

Hypothesis-driven Systematic (proteomics)

Targets Single protein Large numbers of proteins

Known and available (target production usually not part of the

binder generation process)

Usually not known or available (to be provided in an integrated

process)

At least partially characterized Properties unknown

Selection process Number of steps is not an issue Optimized for minimal number of steps

Optimized for lowest failure rate Several percent failure rate is not critical

Resulting binders Individual assay conditions can be specified by end user Standardization (for example, for parallel assays on arrays)

Individual binders not selected or necessarily suited for parallel

application under standardized conditions

Standardized selection and quality control schemes ensure

suitability for systematic approaches (for example, minimized

cross-reactivity)

Large amounts of single binders Small amounts of many binders (for example, for arrays)

Sources Many companies No commercial service available to handle the scale needed

Overall cost Not a central issue Optimized for minimal cost

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a description of the molecular nature of the

binder, the results from quality assurance

and information about suggested applica-

tions and availability of the binder.

Intellectual property. Patent issues may

well influence choices of binders and selec-

tion methods. Intellectual property rights

to binders must be respected. It should be

possible to find a model whereby the inven-

tors of techniques for production of binders

benefit from having their reagents selected

by such a program. The relationship between

an open access resource and commercial

activities will doubtless be a topic of future

debate.

Strategies for the long-term production

phase

Clearly, new binders have to be made in very

large numbers. For antibodies, outsourcing

BOX 2 21

VERSUS 20

CENTURY BINDER GENERATION TECHNOLOGIES:

WHY USE IN VITRO–SELECTED AFFINITY REAGENTS?

In vitro recombinant selection systems link genotype and phenotype of the binding molecule. Features are:

• Scope of targets is not limited by the response of the immune system.

• Sequence information is sufficient to archive and recreate (and eventually distribute?) binders, which may avoid the need for a

physical binder repository.

• Multiple mutagenesis-selection

rounds: binders can be matured to

picomolar affinity.

• Truly monoclonal, but can be made

oligo- or polyclonal as desired.

• Functional domains and tags can be

fused for downstream applications,

for example: detection (GFP, alkaline

phosphatase); multimerization to

generate avidity and/or bispecificity

(coiled coils); technology bridging

(Fc fusions⎯access IgG-based

technology, for example, secondary

reagents); tethering (immobilization

tags, for example, for array

generation).

• Generation of fully human antibody

fragments is possible, for example,

for therapy.

• High throughput: standard

selections yield ≥10 binders per

target.

• Avoid use of animals.

Ribosome display

Phage

display

Cellular

display

Binder gene

Binder

SELEX for

aptamers

Protein-

fragment

complementation assay

century

Huge repertoire

binder genes

Table 2 | Validation criteria and methods for proteome binding molecules (adapted from ref. 7)

Approach based on Examples of methods Issues

Antigen (as used for immunization or in vitro

selection)

ELISA, protein array, SPR The antigen used in the selection is not always the

target for later analyses.

Protein target (from natural sources, for example,

cell lysate)

Western blot, IHC Leaves margin of doubt in the absence of unpurified

target as a control.

RNA (plausibility control to compare mRNA and

protein expression levels)

Transcript profiling, in situ hybridization Correlation of mRNA and protein levels is unknown.

Genetics (use genetic mutants or recombinant

constructs to manipulate target in a defined way)

Transgenesis, RNAi, GFP fusions for comparison

with IHC

Good validation if consistent with observed behavior

of binder, for example, decreased binding upon RNAi

knockdown of target.

DNA (use sequence information on target) Bioinformatic analysis using predictive algorithms Validation of localization in the absence or presence

of transmembrane regions, localization signals and

others

Epitope (compare results from two or more

different binders to different areas, for example,

PrESTs, of same target)

ELISA, protein array, western blot, IHC Mutual validation of binders if corresponding

binding behavior is detected.

IHC, immunohistochemistry; SPR, surface plasmon resonance.

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to multiple sites can be considered. After

adequate assessment, existing binders from

the research community and commercial

suppliers can also be incorporated. However,

quality control and characterization should

be retained within the consortium to ensure

standardization. When trying to match the

available funding with the task of generat-

ing more than 100,000 reagents, it becomes

clear that new solutions and technical opti-

mizations must be found. High-throughput

approaches must be adopted at all levels,

from protein expression and binder produc-

tion to multiplexed assays and multiparam-

eter tests

, and will be an integral part of any

design of a binder-generation pipeline

Networking in and beyond Europe

Other initiatives in the area of affin-

ity reagents are the US National Cancer

Institute proteome reagents program,

focused mainly on cancer-related mono-

clonals

(http://proteomics.cancer.gov),

the HUPO antibody initiative (http://

www.hupo.org/research/hai/) and the

Swedish human proteome atlas project

(http://www.proteinatlas.org). Antibody

Factory (http://www.antibody-factory.de),

is a German national initiative to develop

high-throughput recombinant antibody

methods

35,38

. Additionally, small-molecule

library initiatives have as their ultimate goal

generating ligands for every human protein

function as anticipated by the US National

Institutes of Health Molecular Libraries

Screening Network

(http://nihroadmap.

nih.gov/molecularlibraries/), Germany’s

ChemBioNet (http://www.chembionet.

de/) and the new Spanish ChemBioBank

(http://www.pcb.ub.es/chembiobank/). At

the moment, these initiatives are indepen-

dent and in some cases complementary;

however, given the scale of the problem, it

would seem that in the future, the only way

to tackle the task will be through coordina-

tion of activities.

Meanwhile, individual researchers have

a critical role to play in shaping a resource

that will eventually be theirs to use and

they are expected to contribute to the effort

by their comments and experience. The

ProteomeBinders consortium welcomes

comments and discussion from the wider

community, via the website (http://www.

proteomebinders.org) and participation in

annual meetings (the next open workshop

is in Alpbach, Austria, March 13–16 2007;

details from oda.stoevesandt@bbsrc.ac.uk).

Note: Supplementary information is available on the

Nature Methods website.

ACKNOWLEDGMENTS

This article is dedicated to the memory of our

consortium partner Jane Steel who tragically died

last summer.

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Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

The Wellcome

Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Vrije Universiteit Brussels, B-1050 Brussels, Belgium.

Royal Institute of Technology, AlbaNova University

Center, SE-106 91 Stockholm, Sweden.

University of Zurich, Department of Biochemistry, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Medical Faculty

University of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia.

University of Konstanz, Department of Chemistry, D-78457 Konstanz, Germany.

European Molecular

Biology Laboratory Monterotondo, Via Ramarini 32, Monterotondo-Scalo 00015, Italy.

Laboratoire Bordelais de Recherche en Informatique, 33405 Talence Cedex, France.

Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany.

Corrigendum: ProteomeBinders: planning a European resource of

affinity reagents for analysis of the human proteome

Michael J Taussig, Oda Stoevesandt, Carl A K Borrebaeck, Andrew R Bradbury, Dolores Cahill, Christian Cambillau, Antoine de Daruvar,

Stefan Dübel, Jutta Eichler, Ronald Frank, Toby J Gibson, David Gloriam, Larry Gold, Friedrich W Herberg, Henning Hermjakob,

Jörg D Hoheisel, Thomas O Joos, Olli Kallioniemi, Manfred Koegl, Zoltán Konthur, Bernhard Korn, Elisabeth Kremmer, Sylvia Krobitsch,

Ulf Landegren, Silvère van der Maarel, John McCafferty, Serge Muyldermans, Per-Åke Nygren, Sandrine Palcy, Andreas Plückthun,

Bojan Polic, Michael Przybylski, Petri Saviranta, Alan Sawyer, David J Sherman, Arne Skerra, Markus Templin, Marius Ueffing &

Mathias Uhlén

Nat. Methods 4, 13–17 (2007); published online 28 December 2006; corrected after print 18 January 2007.

In the version of the article originally published, Manfred Koegl’s name was misspelled. Additionally, Zoltán Konthur’s affiliation was incor-

rect; it should be Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany. These errors have been corrected in the HTML and

PDF versions of the article.

CORRIGENDUM

Change-makers bring on recombinant antibodies

Article

Jul 2020
NAT METHODS

Vivien A Marx

There’s a growing affinity for recombinant antibodies. Some say it’s also high time for animal-free recombinants.

Ribosome Display Technology: Applications in Disease Diagnosis and Control

Article

Full-text available

Jun 2020

Antibody ribosome display remains one of the most successful in vitro selection technologies for antibodies fifteen years after it was developed. The unique possibility of direct generation of whole proteins, particularly single-chain antibody fragments (scFvs), has facilitated the establishment of this technology as one of the foremost antibody production methods. Ribosome display has become a vital tool for efficient and low-cost production of antibodies for diagnostics due to its advantageous ability to screen large libraries and generate binders of high affinity. The remarkable flexibility of this method enables its applicability to various platforms. This review focuses on the applications of ribosome display technology in biomedical and agricultural fields in the generation of recombinant scFvs for disease diagnostics and control.

Diverse Human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse

Article

Full-text available

Oct 2019
NEW BIOTECHNOL

We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.

A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins

Article

Full-text available

Apr 2019

There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.

Reduction of Animal Sacrifice in Biomedical Science & Research through Alternative Design of Animal Experiments

Article

Full-text available

Mar 2018

Various upcoming techniques can be used in replacement of experiments requiring animal sacrifice or products of animal sacrifice. In many instances these techniques provide more reproducibility and control of parameter, compared to experiments involving animal or animal products. Use of these techniques can avoid the question of the animal sacrifice during experiment and subsequently permission of ethical approval. In silico simulation, informatics, 3D cell culture models, organ-on-chips are some innovative technology which can reduce the number of animals sacrifice. Scientist evolved some innovative culture procedures and production of animal friendly affinity reagents which are free from the product of animal sacrifice. Direct investigation on human body for treatment as well as further research, electronic health record is also helpful in the reduction of animals sacrifice in biomedical investigations. These techniques and strategies of research can be more cost effective as well as more relevant to various issues related to the human health. Some medical blunder has also been reported after the successful testing of drugs on animal’s model. Hence, the reliability of animal experiment in context with human health is questionable. Alternative to animal experiments help to reduce the number of animals required for research up to certain extent but is not able to eliminate the need for animals in research completely. Wisely use of animals in teaching & research is expected and the importance of animal experimentation in futuristic development in life science cannot be ignored.

The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays

Article

Sep 2017
J PROTEOME RES

Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization’s Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ~43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely-mapping peptides of 9 amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA-abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ~2000 proteins. Finally we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

Article

Full-text available

Sep 2017
PLOS ONE

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.

Antibody-Based Technologies for Environmental Biodetection

Chapter

Sep 2015

Integrated Approaches Toward High‐Affinity Artificial Protein Binders Obtained via Computationally Simulated Epitopes for Protein Recognition

Article

Jan 2019
ADV FUNCT MATER

Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys‐Ep1) show better binding properties than those of lower stability (Cys‐Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL−1) and affinity (dissociation constant (Kd) = 5.3 × 10−11m) are achieved using Cys‐Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders. Combination of computational and experimental studies to develop high affinity artificial protein binders by targeting a cancer biomarker. Highly stable and least stable epitopes of neuron specific enolase are determined by molecular dynamic calculations, and they are used as templates for molecular imprinting. Molecular imprints created with stable epitopes are used as high affinity protein binders, while unsuitable epitope candidates are eliminated.

Antibody validation: A view from the mountains

Article

Aug 2018
NEW BIOTECHNOL

Validation of antibodies and other protein binders is a subject of pressing concern for the research community and one which is uppermost in the minds of all who use antibodies as research and diagnostic reagents. Assessing an antibody's fitness for purpose includes accurate ascertainment of its target specificity and suitability for the envisaged task. Moreover, standardised procedures are essential to guarantee sample quality in testing procedures. The problem of defining precise standards for antibody validation has engendered much debate in recent publications and meetings, but gradually a consensus is emerging. At the 8th Alpbach Affinity Proteomics workshop (March 2017), a panel of leaders in the antibody field discussed suggestions which could bring this complex but essential issue a step nearer to a resolution. 'Alpbach recommendations' for best practice include tailoring binder validation processes according to the intended applications and promoting greater transparency in publications and in the information available from commercial antibody developers/providers. A single approach will not fit all applications and end users must ensure that the reported validation holds for their specific use, highlighting the need for adequate training in the fundamentals of antibody characterisation and validation across the user community.

In vitro Selection and Evolution of Functional Proteins by Using Ribosome Display

Article

Full-text available

Jun 1997
P NATL ACAD SCI USA

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 10(8)-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

An in vivo library-versus-library selection of optimized protein- protein interactions

Article

Full-text available

Aug 1999
NAT BIOTECHNOL

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

High-throughput Screening of Surface Displayed Gene Products

Article

Full-text available

May 2001
COMB CHEM HIGH T SCR

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.

Protein detection using proximity-dependent DNA ligation assays

Article

Full-text available

Jun 2002
NAT BIOTECHNOL

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe . The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 10-21 mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

An affibody in complex with a target protein: Structure and coupled folding

Article

Full-text available

Apr 2003
P NATL ACAD SCI USA

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Erratum: ProteomeBinders: Planning a European resource of affinity reagents for analysis of the human proteome (Nature Methods (2007) vol. 4 (13-17))

Article

Feb 2007

Epilogue: A Personal Perspective: Aptamers after 15 Years

Chapter

Jun 2006

Larry Gold

The BeginningThe First PatentCreation of NeXagen and NeXstarDiagnostic ImagingAptamer TherapeuticsAptamer-based Diagnostics at SomaLogicDo Natural Aptamers Exist?Conclusions – SELEX Lessons for Drug DiscoveryAcknowledgmentsReferences

Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase

Article

Sep 1990
SCIENCE

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

Antibody-Ribosome-mRNA (ARM) Complexes as Efficient Selection Particles for in vitro Display and Evolution of Antibody Combining Sites

Article

Jan 1998
NUCLEIC ACIDS RES

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 104–105-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides

Article

Mar 2000
J MOL BIOL

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.