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Advanced Library Generation

Advanced Library Generation. Tobias Knaute / Johannes Zerweck Peptide Arrays JPT Peptide Technologies, Berlin. What´s the Problem?. The peptide library generation for determining antibody / antigen interactions gets complex if many proteins are involved

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Advanced Library Generation

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  1. Advanced Library Generation Tobias Knaute / Johannes Zerweck Peptide Arrays JPT Peptide Technologies, Berlin

  2. What´sthe Problem? • The peptidelibrarygenerationfordeterminingantibody / antigeninteractionsgetscomplexifmanyproteinsareinvolved • Post-translationalmodificationsincreasecomplexityevenmore • Numberofpeptidesis limited due to experimental setup (max. 7000 peptides per experiment) • Problem: Howtogeneratethemostefficientlibraryconcerningcoverageandcontent

  3. Conventional MethodFixed Overlap C N Overlap = Length - Offset Generation of overlapping peptides based on primary structure MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK ... MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK ... • PRO: • Easy to understand & implement • Sufficientfor simple screeningsandepitopemappings • CON: • Insufficientformanyhomologoussequences • Couldleadtobloatedpeptidelists • Coverage not adjustable

  4. AdvancedMethodDynamicalOverlap • Algorithmenabling optimal coverageofproteinsequencesbypeptidelibraries • Task 1: find optimumbetweennumberofpeptidesandcoverage • Task 2: generatepeptide-score accordingtofrequencyofmotifs (e.g. in otherhomologoussequences) • Task 3: abilitytoinjectfixedsequences (e.g. frompreviousprojects) • Task 4: abilitytoadjustcoverageand/ornumberofpeptidestospecificneeds

  5. Example Protein 1 Protein 2 Task: - 2 homologoussequences - makepeptidescan - peptidelength 15 - maximumoverlap11 - makesureeveryresidueiscoveredbyat least 3 peptides

  6. Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 1 Peptides Protein 1 Protein 2

  7. Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 2 Peptides Protein 1 Protein 2

  8. Example - Convential Scan Convential scan Fixed overlap 15/11 All individual peptides, mapped to protein 1  Several peptides from protein 2 fit into sequence of protein 1 Number of peptides covering specific amino acid in protein sequence  amino acid coverage Peptides 4 5 6 7 8 7 7 7 5 4 Protein 1 Protein 2 Redundancy introduced by homology.

  9. Example - DynamicalOverlap All peptides, mapped to protein 1  Redundant peptides are rejected by algorithm overlap 8 Amino acid coverage is optimized Peptides 3 3 4 4 4 4 4 4 3 Protein 1 Protein 2 Overlap adjustable by coverage constraint.

  10. Case Study: HIV1 ENV gp16024 Sequencesof Group M Subtype B Task: Minimum coverage: 3 peptides/residue Length: 15 Max. overlap: 11 highly conserved region highly variable region 3283 peptides needed for traditional method 2303 peptidesneeded for advanced method

  11. Case Study: HIV1 ENV gp160ResidueCoverageMap Conventional 3283 peptides mean density: 6.7 peptides/residue Advanced 2303 peptides meandensity: 3.7 peptides/residue

  12. Summary • Algorithmtobuild optimal librariesdeveloped • Coverage / numberofpeptidescanbeadjusted • Elimination of redundant motifs • Addressesdiversity, bestsuitedforproteome-widesequencealignments in complexlibraries • ApplicabletoPepStarTMmicroarrays, PepTrackTM Libraries, PepMixTMpeptidepools ... • Based on theinputofproteinsequences, JPT will develop an optimizedlibraryforseroprofilingor immune monitoringexperiments

  13. JPT Peptide Technologies

  14. History 1994 Jerini Spin-Off from Charité Clinics Berlin 2002 Jerini Peptide Technologies - Business Unit 2004 JPT Peptide Technologies GmbH 2005 JPT Inc. in Boston, MA, USA 2006 New JPT Facilities in Berlin 2009 AcquisitionbyBioNTech AG 2011 50 Employees, 13 PhD‘s Offices & Reps Berlin, Boston, Denver, Brussels 1500 sqm wet lab and 1200 sqm office space Capabilities for organic & biological laboratory work Approved S2 lab for infectious patient samples Cleanroom microarray capabilities Business develoment & marketing History, Quality Assurance & Business Model • Quality Assurance • 2002 Quality Management System • 2004 DIN ISO 9001:2000 Certification • Since 2008 GCLP Compliance Audits (NIH, IAVI, BCM, Cellmedica, Genticel...) • 2010 DIN ISO 9001:2008 Certification • Business Model http://jpt.com http://shop.jpt.com http://rnd.jpt.com

  15. Major Application Fields of Peptides Immunology Vaccine development B- and T-cell epitope discovery Diagnostic tools for infectious, autoimmune diseases, allergies & cancer Immune monitoring & immune therapies Proteomics & Enzyme Profiling • Enzyme profiling and protein/protein interaction studies • Quantification of protein expression levels • Biomarker discovery Drug Discovery • Biomedical screening • Peptide therapeutics • SAR, structure activity relationship studies

  16. Feelfreetodiscussyourspecificneedswithourexperts ! JPT Peptide Technologies GmbHVolmerstrasse 512489 Berlin, GermanyT +49-30-6392-5500X +49-30-6392-5501info@jpt.com www.jpt.com • > http://jpt.com • for Custom Products & Services- > http://rnd.jpt.com • for Peptide Related Collaboration Projects • - > http://shop.jpt.com • for Catalog Products

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