ebi European Bioinformatics Institute zahnd-2007-1 A designed ankyrin repeat protein evolved to picomolar affinity to Her2. in vitro ribosome display ribosome display nucleotide sequence nucleotide sequence identification sequence cloning Zahnd C., Wyler E., Schwenk JM., Steiner D., Lawrence MC., McKern NM., Pecorari F., Ward CW., Joos TO., Pluckthun A. J. Mol. Biol. (0022-2836) 2007 3 zahnd-2007-2 A designed ankyrin repeat protein evolved to picomolar affinity to Her2. ecolx Escherichia coli cDNA expression cDNA expression predetermined predetermined participant Zahnd C., Wyler E., Schwenk JM., Steiner D., Lawrence MC., McKern NM., Pecorari F., Ward CW., Joos TO., Pluckthun A. J. Mol. Biol. (0022-2836) 2007 Vector pAT224, Genbank accession number AY327139 zahnd-2007-3 A designed ankyrin repeat protein evolved to picomolar affinity to Her2. in vitro affinity chrom affinity chromatography technology Affinity purification weight by comassie molecular weight estimation by coomasie staining Zahnd C., Wyler E., Schwenk JM., Steiner D., Lawrence MC., McKern NM., Pecorari F., Ward CW., Joos TO., Pluckthun A. J. Mol. Biol. (0022-2836) 2007 zahnd-2007-4 A designed ankyrin repeat protein evolved to picomolar affinity to Her2. in vitro spr surface plasmon resonance BIAcore(r) Optical biosensor BIAcore(r) Optical biosensor predetermined predetermined participant Zahnd C., Wyler E., Schwenk JM., Steiner D., Lawrence MC., McKern NM., Pecorari F., Ward CW., Joos TO., Pluckthun A. J. Mol. Biol. (0022-2836) 2007 Construction of point mutants: The mutations were introduced by a PCR-based approach. Two fragments were amplified overlapping in the region of the desired mutation. In a second PCR reaction with outer primers only, the fragments were joined to yield the mutated sequence. Clone G3-D was constructed with a single PCR reaction using a long reverse primer, as the mutation was near to the C terminus. DNA of all clones was isolated from agarose gels and cloned via BamHI and HindIII into pQE-30 (Qiagen). Plasmids were prepared, sequenced and the mass of the expressed and purified clones was verified with mass spectrometry (MALDI-TOF). Biacore 3000 (Biacore) zahnd-2007-5 A designed ankyrin repeat protein evolved to picomolar affinity to Her2. in vitro sandwich immunoassay sandwich immunoassay antibody detection identification by antibody Zahnd C., Wyler E., Schwenk JM., Steiner D., Lawrence MC., McKern NM., Pecorari F., Ward CW., Joos TO., Pluckthun A. J. Mol. Biol. (0022-2836) 2007 Each sample was immobilized on Luminex beads of distinguishable color-code according to the manufacturer's protocol (Luminex-Corp., Austin, Tx, USA). h10-2-g3_2jab Designed Ankyrin Repeat Protein (DARPin) binding human epidermal growth factor receptor (Her2) darpin ankyrin repeat ankyrin scaffold in vitro MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYLATAHGHLEIVEVLLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKTAFDISIGNGNEDLAEILQKLN erbb2_human Receptor tyrosine-protein kinase erbB-2 precursor ERBB2 HER2 NEU NGL protein protein human Homo sapiens MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV sp185_ab sp185 antibody from BenderMed Systems mab monoclonal antibody mouse Mus musculus BenderMed Systems lib_screen Library screening experiment producing h10-2-g3 / 2jab a designed ankyrin repeat (DARPin) binding human epidermal growth factor receptor 2 (Her2) 2 h10-2-g3_2jab 4 affinity reagent protein affinity reagent par generation product generation product prey prey ecolx Escherichia coli erbb2_human 6 par target protein affinity reagent target bait bait biotin tag Biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined sequence position false ecd Extracellular domain extracellular domain extracellular domain HPE tele-phosphohistidine histidine-N1'-phosphate 1'-phospho-L-histidine histidine-N(epsilon)-phosphate [H:po_e] tau-phosphohistidine undetermined undetermined sequence position undetermined undetermined sequence position false unknown Unknown BenderMed Systems, Vienna, Austria physical interaction physical interaction aggregation h10-2-g3_2jab_purif Purification of expressed h10-2-g3 / 2jab DARPin binding Her2 10 h10-2-g3_2jab 4 par target protein affinity reagent target generation product generation product ecolx Escherichia coli purified physical interaction physical interaction aggregation darpin_affinity Affinity determination of DARPins including h10-2-g3_2jab 13 erbb2_human 6 par target protein affinity reagent target bait bait biotin tag Biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined sequence position false ecd Extracellular domain extracellular domain extracellular domain HPE tele-phosphohistidine histidine-N1'-phosphate 1'-phospho-L-histidine histidine-N(epsilon)-phosphate [H:po_e] tau-phosphohistidine undetermined undetermined sequence position undetermined undetermined sequence position false unknown Unknown BenderMed Systems, Vienna, Austria h10-2-g3_2jab 4 affinity reagent protein affinity reagent par prey prey ecolx Escherichia coli direct interaction direct interaction 13 13 13 Table 1, Figure 2 h10-2-g3_2jab_expr-1 Expression of H10-2-G3 / 2jab, a high affinity binder for Her2 19 h10-2-g3_2jab 4 unspecified role unspecified role intermediate product intermediate product in vitro unpurified physical interaction physical interaction aggregation h10-2-g3_sensitivity Sensitivity determination of DARPins including h10-2-g3_2jab 22 sp185_ab 24 unspecified role unspecified role secondary par secondary protein affinity reagent detection antibody BenderMed Systems, Vienna, Austria h10-2-g3_2jab 4 affinity reagent protein affinity reagent par bait bait n-terminal region N-terminal region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined sequence position false n-terminal region N-terminal fusion protein avi-pD fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined sequence position false ecolx Escherichia coli erbb2_human 6 par target protein affinity reagent target prey prey ecd Extracellular domain extracellular domain extracellular domain HPE tele-phosphohistidine histidine-N1'-phosphate 1'-phospho-L-histidine histidine-N(epsilon)-phosphate [H:po_e] tau-phosphohistidine undetermined undetermined sequence position undetermined undetermined sequence position false unknown Unknown BenderMed Systems, Vienna, Austria direct interaction direct interaction Figure 6a Concentration H10-2-G3 (pg/,ml) : Signal/Noise ratio; 2:2, 9:3, 40:9, 200:60, 1000:200, 5000:800, 20000:3000