Download PepIDENT — bio-peptide Library Expression System for Epitope

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PepIDENT — bio-peptide Library Expression System
for Epitope Screening and the Development of
Focussed Diagnostic Arrays
Preface
Phage display libraries are the most known type of completely random one peptide variant libraries.
Other types of one peptide variant libraries are covering more specific pattern of peptide variants
which need to be calculated and synthesized accordingly.
But there are very different types of peptide libraries which are designed like the consecutively ordered pages of a book. The book represents the protein, the pages of the book the peptides. The
library design therefore is like walking through the sequence of at least one protein for e.g. the diagnostics of autoimmune diseases and heat shock protein involvement. Other more complex schemes
of ―protein walking‖ bio-peptide libraries can be generated for immunological infectious disease research covering the whole genomes of viruses with bioPeptides (Peptidome) for example.
The pattern design can be explained like in case the recognition of an epitope by an antibody paratope needs two parts of text located on two different pages e.g. the bottom lines of the one page and
the top lines of the next page the specific recognition can only be realized if there are overlapping
schemes of different pages. Bio-Peptide libraries walking along protein sequences in overlapping non
-random schemes can be designed on the basis of gene synthesis. Their handling in analyses are
known from routine molecular biology working protocols e.g. for complex random phage libraries
handling in case the epitope handling is on phages rather than with GST or other carriers.
Preselected functional sub-genomic groups of target proteins can also covered completely by such
types of libraries. This can be done by support of bio-computing and proteomics.
With the methodology described systematic analyses of peptide pattern covering many potential targets and for the assessment of the immune status of different medical indications can be performed
and diagnostic tools developed. Relevant epitopes for autoimmune diseases, infectious diseases and
for the development of immune-therapies of tumors as well as allergens can be identified. Aiming on
the identification of specific epitopes a broad range of applications starting with the development of
new immune diagnostics is possible. Immune diagnostics can be provided in different formats like
micro-titer plates, micro-array based systems, stripes or other matrices suitable for point of care serum diagnostics.
The PepIDENT - technology is suitable for the synthesis of peptide coding sequences (pCDS) creating stable 1:1 stoichiometry in pCDS - coding sequence maintenance libraries. This stoichiometry
can be guaranteed. Once synthesized the sequence maintainance library can be propagated infinitely. Thus we can redeliver to you carrier bound peptides generated by peptide CDS – expression
libraries as long as you need in always the same quality in large amounts without de novo synthesis
just by re-synthesis.
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Analysis and Identification of Specific
Target - Epitope : Antibody - Paratope — Interactions
PepIDENT is a new generation of protein specific bio-peptide library systems. bio-peptides are produced by the ribosomes of E. coli. One important feature is the freedom in design of bio-peptides of
different sizes ranging from standard peptide size to mini-proteins in size.
Originally the first bio-peptide library systems were designed for the identification of serum polyclonal
antibodies. These were directed against individual epitopes of HPV viral oncogene proteins. E7 protein specific bacterial peptide libraries of variable complexity/ diversity were expressing very systematically designed. Starting from this initial epitope mapping systems the identification of epitopes from
all proteins specificities was further developed. The bio-peptides are fused in frame to different possible carrier proteins (e.g. GST) chosen for the desired application.
Later the system was designed specifically for the identification of auto-antigen epitopes and comparing responses between healthy individuals and patients with different auto-immune diseases. The
peptide libraries can be designed for to contain peptides from a series of functionally related proteins
(receptor molecule variants) or even selections of unrelated proteins for example just surface proteins from a defined pathogen. Families of sequence related proteins can also provided as libraries
like peptide libraries of whole proteomes. This feature creates the new discipline of Synthetic Proteomics.
Two developed applications using the current version of the system successfully are:
Epitope mapping for monoclonal antibodies.
This application can identify linear epitopes and epitopes with secondary structures, depending
upon the length of the peptides used. The peptides are synthesized and cloned into an expression vector in which they are fused to the carboxyl terminus of glutathione-S-transferase. The
library is plated out to single colonies and making sure that there are sufficient colonies to cover
the entire library at least four times (e.g. if the library contains twenty peptides at least eighty
colonies are needed). The colonies are then transferred to a nitrocellulose filter, grown under
expression-inducing conditions and lysed in situ. The filters are incubated with the monoclonal
antibody and if necessary a secondary antibody before visualizing with a chromogenic substrate.
Positive colonies are picked from the master plate and the sequence of the insert is determined
to identify the peptide. The positive colonies should all contain peptide sequences with the consensus recognized by the antibody.
Mapping immune responses directly in sera.
This application recognizes similar epitopes to the example above. However the peptides are
expressed as an expression library that is linked to either the pIII or pVIII protein of M13 and displayed on the surface of phages.
The fusion proteins are expressed from phagemid vectors that are inducible and displayed using
a helper phage system. This means that the library can be amplified without loss of complexity.
The library is grown up so that the peptides are expressed in the phage surface.
The sera of interest are used to coat microtiter plates. The phages are added to the wells and
allowed to bind to the antibodies in the serum. The bound phages are eluted and used to infect
host bacteria that are then plated out onto selective medium and also grown up to produce a library that can be used for further rounds of panning (although this is not usually necessary). The
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resulting plasmids are then sequenced to determine the sequence of the inserted peptides.
This procedure not only determines the epitopes present but will also give a quantitative estimation of that is not possible with other methods since the number of phages with each sequence is
proportional to the amount of antibody present. The only requirement is to sequence a sufficient
number of clones in order to attain a statistically valid result.
Additional refinements of the procedure allow different classes of antibody to be determined directly in serum.
Using this system there is no limit to the number of sera that can be screened as the library can
be re-grown from a stock when required. A single culture can generate sufficient phages for a
large number of assays.
Another application is to look for cross-reactivity between different related proteins, for example
to immunize with one protein and pan using a related protein. However, such experiments require pre-immune sera as controls.
Technical remarks:
The bio-peptides can be designed by the customer for a single or variable size without size limitation
in a physiologically acceptable range (e.g. 5 aa to maybe 50 amid acids for even for very big epitopes or scrambled interaction points).
Therefore to differentiate from its chemical counterparts we call these bio-peptides because of having
advantageous features.
Each of the E. coli clones is expressing one single type of the designed bio-peptides of the whole
library diversity fused to a carrier protein of your choice.
Carrier proteins can be designed for:
(1)
binding the library molecules to a surface specificly or non-specifically for presenting
the epitopes to environment of the assay or can
Glutathione-S-Transferase - Gst-Tag
Phage P1 - gIII/ gVIII
ProteinA
Streptavidine
c-Myc-tag
T7-Tag
Arg-Tag
Flag-Tag
Strep-Tag
V5-Peptide-Tag
c-Myc-tag
S-Tag
HAT-tag
Calmodulin-binding peptide
Chitin, Cellulose or Maltose -binding peptides/ protein tags
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(2)
use the specific detection properties of a reporter gene like fluorescence, luminescence
or colorimetric enzyme assay.
GFP, RFP, YFP, Luciferase, Chloramphenicol Acetyltransferase—CAT-Assay,
HR-Peroxidase etc.
(3)
... other favourable custom made features
Using different bio-peptide—carrier protein library systems you can:
Screening for specific interaction of polyclonal serum antibody paratopes with individual biopeptide-epitopes for the assessment of the immune status of patients in:
autoimmune diseases
cancer autoimmunogenic reactions etc.
infectious diseases
allergic reactions
Screening different bio-peptide-epitope clones against the specificities of monoclonal antibody paratopes origin of different origin e.g. quality control antibody production
Screening/ Scanning for Protein-Protein Interaction – using bio-peptide - reporter - carrier
fusions produced by individual E.coli - clones
Assaying the interaction of proteins on cell surfaces other than E. coli
Assaying the interaction of bio-peptides with proteins bound on surfaces
Assaying the interaction of bio-peptides derived from specific proteins bound to surfaces
with bio-peptide-reporter-fusions of other proteins forming complexes
... other, customer defined applications
Technology: In a first step during the primary synthesis the whole library is based on one to a few
single synthetic dsDNA molecules comprising all peptide CDS entities of a single protein for one bioassay, several proteins in one bio-assay or variants of one protein for bio-assays.
In a second step for its expression, the peptides typically are fused to different possible carrier proteins providing either specific binding properties to a technical or to a cellular surface for setting up a
bio-assay. Or it can be fused to a reporter protein as well as to other proteins of desired functions.
Peptide expression is directed via an inducible expression vector system. This provides the advantageous not to subject bio-peptides towards any selection in a expression bias. Un-induced plasmids
are handled neutral in the early growth stages of the bacterial culture. Aliquots of the primary expression library can be used to inoculate many secondary expression cultures. If there is no expression bias of peptides reducing the diversity/ complexity of your library then one can use the primary
expression library for infinite inoculation cycles and late stage induction. If there is an expression bias
in your bio-peptides expression you can re-order either the bio-peptides ready to clone PepIDENT –
Kit or the primary expression library.
The PepIDENT—Technology can be designed for at least four different variant assay types:
(1) bio-pep - simple filter screening assays and random distribution of bio-peptide E. coli clones
(2) dot-ELISA-Filter Assays preselected positional control of E. coli clones or purified bio-peptides
(3) Microtiter -Well-ELISA-Plates preselected with positional control and purified bio-peptides
(4) PepID-Diagnostic-MicroArrays preselected positional control of purified bio-peptides
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Fig.1-1. Functional bio-peptide-Library Maintainance and Expression System. Library maintainance vector pPepCDS and bio-peptide—expression vector pEPX1. Each containing peptidecoding sequences (PepCDS). pPepCDS plasmids contain all single bio-peptide-CDSs of a protein
peptide library design in a compact formate. In pEPX1 the bio-peptides are individually fused to carrier proteins of desired properties like GST-Tag (Gluthatione-S-Transferase). This bio-peptide library will be provided custom made for any individual proteins desired.
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The individual types of libraries differ in its design and in its ideal type of deployment.
Peptide-epitope random access libraries are as complex as they are designed, ranging from low
complexity of one protein library to very high complexities with multiple proteins. Complexity is the
choice of the experimentator. The way how to make the libraries is patented. Each contains an individual carrier protein. Carrier proteins can be easily exchanged. The carrier proteins are fused with
individual epitopes and expressed by individual E. coli clones. Individually arranged in an ordered
well format the assay can be designed as a DOT–ELISA. In any format each of the clones is just determined by the specificity of the epitope expressed. Expression of the epitopes is inducible which
has some advantages. The PepIDENT-technology should be applicable also with glycosylated epitopes. This could be achieved by use of glycosylating expression systems in vivo or by selectively
glycosylating the membrane bound epitopes with Glycosyl-Transferases in vitro. This has not be
Protein:Protein Interaction
Minimal Sequences of
Antibody:Epitope
Recognition& Binding
Functional Diagnostics for
Rationally Directed
Evidence Based
Individually Applied
Personalized Medicine
Fig.1-2. Systematic and flexible designs of low to high resolution pattern of protein-sub-sequence peptides for e.g.
Autoimmune Epitope screenings can be made Online at ATG. Protein sequences are dissected online and all bio-peptide
entities are cloned with its individual coding sequence into the library maintainance vector pPepCDS. From this maintainance
vector all coding sequences can be regenerated in a 1:1 stoichometry if desired and neccessary. The inducible vector pEPX1
is used for expression cloning of bio-peptides and use in the bioAssay systems they are designed for.
Advantages of Ribosomal - PepID - Peptides Relative to Chemical Synthesis
First Synthesis of each library is in one piece - peptide - sizes can be precisely predefined
Very systematic peptide pattern designable without sequence preference to equal cost
All sizes from smaller peptides to larger entities (Mini-)Proteins are possible (e.g. 5 – 50 aa)
Defined peptides of high biological quality - no side reactions like in Peptide Chemistry
1:1 Stoichiometry of different Peptides can be guaranteed
Smaller libraries size compared to random libraries with defined low complexity
NO irrelevant Peptides NO empty vectors and comparable very low background in analysis
NO loss of complexity through amplification.
Different carrier proteins (e.g. GST-tags) can flexible be applied to one and the same library
Once synthesized the library can be propagated in a number of different forms!
Deployment from Random Access Libraries to Epitope MicroArrays
Ideal e.g. for AutoImmune Disease - AutoAntiBody – Individual Pattern Screenings
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Demonstration of the specificity of primary structure binding
of AB1 and AB2 by clear positives (boxed in light gray and
grey on filter assays. Schematically on the right specific affinity binding of epitopes presenting clones marked in Serum
Antibodies sAB are labelled in red, green and yellow.
Fig.2. Epitope - Random Access Expression Libraries. Peptide - CDS - clones were fused to the
GST - carrier- protein gene. Individual clones were plated by random distribution on agar plates.
shown yet but should be feasible in an appropriate experimental design.
The clones are plated randomly on the surface of a Petry-dish and subsequently are transferred to a
suitable reaction membrane. Before the antibody specific reaction identified the clones it is not clear
which of the clone-specific expressed peptides are to be allocated to which antibody. This becomes
immediately clear by identifying the positive clones applying antibodies for binding reactions (e.g.
from serum) and subsequently sequencing the peptide coding DNA sequences of clones identified
positive for binding antibodies specificly. Therefore access to the positive clones is random but its
specificity mediates the correct epitope-paratope interaction for identification of the positive binders.
For basic R&D work the most simple epitope peptide random access type of libraries are a fast, reliable, convenient in use and are a comparably cheap way for epitope mappig and thus for getting
valuable results.
Fig. 3: PepIDENT : Peptide MICRO - ARRAY – library – formats deposited with the Top-Spot Technology Diagnostic devices for the identification of autoimmune- and allergene - antibodies, can be
manufactured by use of the PepIDENT-technology.
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In contrast to Peptide Random Access Libraries mentioned before the MicroArray based PepIDENT
- technology provides additional positional information by selecting each of the possible clones in
advance and positioning it on a MicroArray for allocating each of the peptides to a positional code.
This helps to identify relevant epitopes immediately without the need for sequencing.
Patient related diagnostic PepID applications do have a much higher need for fast and easy
readout of the diagnostic information. This is feasible by placing validated PepIDENT —
epitopes on MicroArrays and qualifies the PepIDENT – technology for having the potential to
create products for the diagnostic market. In order to fully exploit its potential these types of
PepID—devices need to be standardized for placing it cost-effectively on the market.
In turn this generates the advantage to identify pattern of polyclonal antibody - allergen and autoimmune reactions directed against known targeted proteins direct in serum.
But in addition also new target proteins can be identified in screening projects.
Especially for the characterization of autoimmune or allergen antibody pattern the identification of
relevant epitopes is of great diagnostic value in case to case studies and provides the basis for the
assessment of individualized therapeutic schemes.
Screening programs accompanying clinical trials or just point of care diagnostics close to the patient
directly at the doctor's location e.g. the clinic can be performed with micro-array based libraries.
With PepIDENT – Plasmid/ Phagemide Libraries coding CDS sub-units can be generated coding for
small core secondary structures or even to smallest linear epitopes. But also coding sequence pattern can be created which are covering larger protein structures e.g. on the domain level still capable
for folding a domain tertiary core structure. In protein specific sub-sequence libraries binding pattern
of different sizes can be systematically combined and arranged in overlapping schemes on different
possible binding levels for the analysis of the binding mode. Protein derived libraries can be provided
e.g. pEPX1 – Expression Vector with a Gluththione-S-Transferase - GST - Tag or phage displayderivative Vectors like pEpMAP-Vector-Systems.
For precise CDR = complementarity determining region characterization Alanine Scanning Mutagenesis (ASM) provides the highest resolution of epitope characterization possible. Libraries substituting each of the amino acids of an identified epitope with Alanine can be easily designed and applied
to the peptide library binding assays for exactly identifying the amino acids involved in binding.
With the PepIDENT– BINDING - ASSAY differentiation between different modes of interactions or
unraveling minimal binding structures for its linear specificity or higher order binding is possible. For
clear results of characterization and identification it is recommended to combine certain pattern in an
experimental strategy.
High resolution IDENTification of specific complementarity determining regions of: CDR - Paratope:
Epitope Interaction – Interfaces can be achieved by the Alanine Scanning Mutagenesis screening
procedure substituting functional amino acids - groups.
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READY-TO-USE - EPITOPE BASED LIBRARIES FOR - DO-IT-YOURSELF - ANALYSES
Identification, Characterization and Validation of Antibody Binding Pattern
Mono- and Poly-Clonal Antibodies Directly with Patient Serum
Precise Identification of Immunogenic Determinants in Proteins
Lowest Background in Analyses Because of Adaptive Complexity
DEVELOPMENT of different IMMUNO THERAPIES and VACCINATION for Epitope Specific
Modulation of Immuno-Cell Responses in
Autoimmune, Desensitization, Vaccine Development
Allergology, Desensitization
Infectious Diseases - Epitope Based Vaccine Development
Cancer Immunotherapies
High Resolution EPITOPE IDENTIFICATION of Sub-Sequence Antibody Binding Sites
Diagnostics of general IMMUNERESPONSE in Serum
B-Cell Responses (HPV/ E6/ E7)
T-Cell Responses
CROSS - Reactivity Measurement of Immunizations (HPV 16/18)
AutoIMMUNE Diseases
Rheumatoide Artheriosclerosis
Arthritis
Diabetes
Neurodermitis …
ALLERGIC Reactions - IDENTIFICATION of Allergen-Epitopes
INFECTIOUS Diseases – Influenca, HIV, HPV, HCV, Malaria …..
Thus PepID-Applications are in general powerful for both, testing patients for
their current immunostatus or for immunologic patho-pattern and the development of therapeutic agents.
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Fig.4. Future Patent Protection of Antibodies. Detailled description of the Antibody targets primary
sequences, secondary structures or scrambled epitopes is recommended for patenting new specificities. The epitope as well as the correlated paratope can be described in detail with the methodology
recommended in this brochure.
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