3401 YCHPQFCG(4) HCHPQFCS(3) LCHPQFCG(2) DCHPQFCS(2) NCHPQFCP(1) DCHPQFCR(1) DCHPQFCV(1) HPQ 7 Phage display (common panning) 3 PMID:8508957 Streptavidin Biotin Not determined. Not determined. TN2 phage display library (XCX4CX) Library fractionation involved a 2-h incubation of phage with target, ten rapid washes with PBS containing 0.1% Tween-20 (to remove unbound phage), and stepwise washes with citrate buffers of pH 7, 6, and 5. Bound phage were removed from target-coated wells by the addition of pH 2 citrate-buffered elution solution for 10 min followed by immediate neutralization with Tris pH 8 buffer to a final concentration of 250 mM Tris. A portion of the phage eluted at pH 2 was used to grow a fresh stock of display phage which was then further fractionated. This sequence of events constitutes a single round of enrichment and amplification. 3402 LCYGGFCD(2) SCYGAFCR(1) VCYGAFCR(1) RCPTAGCD(1) NCYGAFCH(1) LCYGGFCP(1) 6 Phage display (common panning) 3 PMID:8508957 Anti-beta-endorphin monoclonal antibody 3-E7 Beta-endorphin Not determined. Not determined. TN2 phage display library (XCX4CX) Library fractionation involved a 2-h incubation of phage with target, ten rapid washes with PBS containing 0.1% Tween-20 (to remove unbound phage), and stepwise washes with citrate buffers of pH 7, 6, and 5. Bound phage were removed from target-coated wells by the addition of pH 2 citrate-buffered elution solution for 10 min followed by immediate neutralization with Tris pH 8 buffer to a final concentration of 250 mM Tris. A portion of the phage eluted at pH 2 was used to grow a fresh stock of display phage which was then further fractionated. This sequence of events constitutes a single round of enrichment and amplification. 3403 WTRSDHRIQ(2) WTRDQHQIH(1) WTLREHDFH(1) WQITQHKLQ(1) WTLQHHRVV(1) WTLGEHTLI(1) WRLSDHRMV(1) WTIKDHQLL(1) WKLSEHRMA(1) WSLGQHRIF(1) HGKHTHKVG(1) HGDKHKHRG(1) KPHQHKVHK(1) 13 Phage display (common panning) 3 PMID:10211822 Protein A Not determined. Not determined. Not determined. J404 phage display library (X9) Phage were selected by direct binding to the protein A matrix in the absence of antiserum for three rounds. 3404 LTFQGLP SHRGPSF GHWHFQE 3 Phage display (common panning) 3 PMID:11314263 Goat anti-human IgM polyclonal antibody Human immunoglobulin M (IgM) Not determined. Not determined. Ph.D.-7 phage display library (X7) 3405 GLTFQLHHQMRP LSFQNTVPPWAT GLMFQHPTLVPW KLNFQGSPYEPR LQFQNIESPLSP LQFQSLPDFTYS GLTFNNPAPFAG SLSFQTLKPLHP DHFSFQGLSSTG SHFSFQTGRSAL SHWSFQDRLSAI GHWGFQKDDPPW HSGDWLTNSPRV 13 Phage display (common panning) 3 PMID:11314263 Goat anti-human IgM polyclonal antibody Human immunoglobulin M (IgM) Not determined. Not determined. Ph.D.-12 phage display library (X12) 3406 GQSEKHL HGGVRLY SLSKWSF KIAVIST TDNTKPK TNWRTIN VSRDTPQ 7 Phage display (common panning) 3 PMID:30854940 Endothelial Progenitor Cell (EPC) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) 3407 SFKIPYHYDSGQ TIPRAPSPANTY NRPDSAQFWLHH 3 Phage display (subtractive panning) 3 PMID:30854940 Endothelial Progenitor Cell (EPC) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) The positive selection was performed against Endothelial Progenitor Cells (EPCs) just after the negative selection against platelets, and the eluted phage clones were amplified only after these steps were completed. 3408 CPLDLRSPC[27.5 ± 0.5] 1 Phage display (common panning) 3 PMID:30867462 B1 peptide (CMSDWTGG) Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA Binding of the B1.12 peptide to its target B1 was evaluated by phage ELISA. Blocking buffer without B1 peptide was used as negative control. All experiments were done in triplicate and the binding of the phage clones to the B1 target was calculated according to the following formula: binding = OD phage/OD control × 100, where OD control and OD phage represent the OD410 nm values of phage binding with negative control and with B1 peptide, respectively. The binding value was regenerated from Figure 1a and shown. A solution of 1 mg/mL of the B1 peptide in 100 mM NaHCO3 (pH 8.6) was prepared and 100 μL was coated on a 96-well plate and incubated overnight at 4 °C. The coating solution was poured off and blocking buffer (skimmed milk at 5 mg/mL in 100 mM NaHCO3) was added for 1 hour at 4 °C. After washing with 0.1% (v/v) Tween-20/TBS, 10 μL (2e11 plaque forming units, pfu) of original library, Ph.D™-C7C Phage Display Peptide (New England Biolabs), were diluted in 100 μL TBST and added to the plate well for 2 hours at room temperature with gentle agitation. After ten washes with 0.1% TBST, the bound phages were eluted with 0.2 M glycine-HCl (pH 2.2) and neutralized with 1 M Tris-HCl (pH 9.1). Eluted phages were amplified in 20 mL LB inoculated with the E. coli ER2738 strain, purified by PEG/NaCl precipitation, and titrated as described in the NEB standard protocol to be used for the next round of selection. The two next rounds of selection were performed under more stringent conditions as the concentration of Tween-20 was gradually increased (0.3% and 0.5% for the second and third round, respectively) and the incubation time of phages with the target was gradually reduced (1.5 hours and 1 hour in the second and third rounds, respectively). We identified by Phage display a novel peptide called B1.12 (ACPLDLRSPCG) which selectively binds to the extracellular loop (B1) of the latent membrane protein 1 (LMP1) as demonstrated by molecular docking, NMR and ITC. Using an LMP1 expressing cell line, we showed that B1.12 decreased cell viability, and induced G0/G1 cell cycle arrest. In addition, the expression of A20, pAkt, and pNFkb (pRelA536) in C666-1 cells treated with B1.12 decreased compared to the untreated cells. In conclusion, we selected a novel peptide able to bind specifically to the extracellular loop of LMP1 and thus modulate its oncogenic properties. 3409 GTTVSQFLS(2) VSPSLINEV(1) SVPTLVNLT(1) VVPVVVRTS(1) VAPVVVVLF(1) VSPVVVVEV(1) NFPVVVWDV(1) WAPLVVMDW(1) GVMPEVVTE(1) VEVVQLHVS(1) VEGVIILGS(1) LEPVTISAG(1) LLEPILEKR(1) GPPVLMGILI(1) FRLVTLSLG(1) ARTVWGFAG(1) VVQTVMLIS(1) TVQVLERLA(1) MVVASDVIG(1) EVLVVDKVM(1) SVVLATLKI(1) AVTLAQLRL(1) VMLAYLAVP(1) GVLVSLATS(1) GLTVADSGDL(1) GLYIAESFY(1) GLVIRELRRL(1) GLMVRETLGT(1) GLLISLVAR(1) GLLVSEVWTA(1) VGTLLVSYM(1) GALTVSLRV(1) GALLVFERW(1) WVLAVMEHR(1) ECLVIEELR(1) CRLVVIEHV(1) KLVINELVR(1) VAIRVMVRC(1) FTKIREVLG(1) 39 Phage display (common panning) 5 PMID:12900423 Chymase (EC:3.4.21.39) Not determined. Not determined. Not determined. X9 T7 phage display library An aliquot of the amplified phages (~e9 plaque forming units) were allowed to bind to 100 μl of Ni-NTA-agarose beads for 1 h while rotating gently. Unbound phages were removed by washing 5 times with 1.5 ml of 1 M NaCl, 0.1% Tween 20 in PBS (pH 7.2) and further washed twice in 1.5 ml of PBS and finally resuspended in 270 μl of PBS. A control elution of the phages with 100 μl of 500 mM imidazole concluded that at least 1.0e8 phages were attached to the matrix after washing. rMCP-5 was digested with enterokinase as previously described in the absence of heparin and then subjected to heparin-Sepharose purification. After elution with PBS containing 1.5 M NaCl, heparin was added to a 10:1 mass ratio of heparin to protease. The selection was started by adding ~0.2 μg of protease (~27 nM final concentration) or buffer without protease as a control to the tubes with the resuspended beads. The protease was allowed to digest susceptible phages overnight with gentle agitation. To recover released phages, the supernatant was removed, and the beads were washed in 100 μl of PBS. To ensure that all recovered phages lacked the histidine tag, 15 μl of fresh Ni-NTA-agarose beads were added to the phage suspension, and the mixture was agitated for 15 min followed by centrifugation to recover the supernatant. Ten μl of the supernatant was used to determine the amount of detached phages in each round of selection. The remaining 290 μl of the supernatant was added to a 10-ml culture (A = ~0.6) of Escherichia coli (BLT5615). The bacteria had been induced with 100 μl of 100 mM isopropyl-1-thio-β-D-galactopyranoside 30 min before phage addition to ensure production of the phage capsid protein. After six rounds of selection, 40 plaques were arbitrarily isolated from LB plates. The oligonucleotide inserted into the phage DNA was amplified by PCR, and the randomized sequence was determined by nucleic acid sequencing. 3410 LVTFLSLSA GILFLSSLIS LAVLFRSLL STLFVGSSV VALLWSLVS VMLWVSSAA GQLYSSVLLD VLQRFSMER VMRFVMAIG VVRFLSGLC IVRWLSINV IMTWISVET RCSAFVLES VLLFMGNWV VALFWLQQV VSLFMGIDF VQLYWVVEE SLELWMSMW GVELFFLRM ELRMLFAAF GGLYSYVVME VVVYGGFEI IVVVRYSLF VERWSWVLV WGVSFLSVV DVWFNLLLN EISMYFSLV VRCFMALGY VMMSYTFVRH VPMAWIGYA WEPDRWVSFH GVRFLSFENW MLLRFWAYE WVVWISKEF WVSYTSRLW WWAVVSFVGH YVTFGGLWG SLMFFWARL WDWMPFLAS GWMFYDAILT 40 Phage display (common panning) 5 PMID:17681377 Chymase (EC:3.4.21.39) Not determined. Not determined. Not determined. X9 T7 phage display library An aliquot of the amplified phages (~e9 pfu) was allowed to bind to 100 μl Ni-NTA agarose beads for 1 h while rotating gently at 4 ◦C. Unbound phages were removed by washing ten times with 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, and two subsequent washes with 1.5 ml PBS. The beads were finally resuspended in 1ml PBS. Activated and heparin-sepharose purified mast cell protease 1 was added to the resuspended beads to start the digestion of susceptible peptides. Buffer without protease was used as a control. Protease digestion was allowed to proceed at room temperature over night with gentle agitation. To recover released phages, the Ni-NTA agarose beads were pelleted by quick centrifugation and the supernatant containing the phages was removed. To ensure that all of the released phages were recovered the beads were resuspended in 100 μl PBS (pH 7.2), which was removed and added to the first supernatant after centrifugation. A control elution of the remaining phages still bound to the Ni-NTA agarose beads, using 100μl 100mM imidazole concluded that at least 1.0e8 phages were attached to the matrix during each selection round. To ensure that all phages recovered after the protease digestion step lacked the His6 tag, 15μl fresh Ni-NTA agarose beads were added to the phage suspension and the mixture agitated for 5 min followed by centrifugation to recover the supernatant. Thirty microlitres of the supernatant was used to determine the amount of detached phages in each round of selection. The remaining 1070 μl of the supernatant was added to a 10ml culture (OD~0.6) of E. coli BLT5615. The bacteria had previously been induced to produce the phage capsid protein by adding 100 μl 100m MIPTG 30 min prior to phage addition. Approximately 75 min after phage addition the bacteria lysed. The lysate was centrifuged to remove cell debris and 900 μl of the phage sub-library was added to fresh agarose beads. After binding and washing the sub-library, a new round of selection was started. Following five rounds of selection, ~100 plaques per enzyme were arbitrarily isolated from LB plates and each dissolved in phage extraction buffer (100mM NaCl and 6mM MgSO4 in 20mM Tris–HCl pH 8.0) and vigorously shaken for 30 min in order to disrupt the phages. 3411 GVLFVEASA VSLFLEVQE GVLFLELEGV GGAVLFLEL VVLWSDIGE SVMRWSEVL PVAYAAGGV HMMVVYRNS EVVLRSDWNHH WLAYSEEEV SRFVSFVDD LVSYGEVFL RVLQYLEYSP IAVFLDVYE VARFLDLSF WEVWMEMRT GWDLWQDIS WDVPMLWRDH VVSVFYNQD ISLYLAPIW PFSNFMSLG WVTTYLSGG GWMLRYLAG FLSFWDLAG TYFSFWDLAG TYSVTFTEH WGWLLFREL AWALYVEYG WEAVGYMGW WQVLRFWGM WASVQFFMG WAPWTYLLT IWMFYFSSA GCERFFRWI GGWWLTAYISI WVSLWFWEE WVVYGGWYL WYYFSSYIF PGGWDWGYYVGY 39 Phage display (common panning) 5 PMID:17681377 Mast cell protease 4 (EC:3.4.21.-), mMCP-4 Not determined. Not determined. Not determined. X9 T7 phage display library An aliquot of the amplified phages (~e9 pfu) was allowed to bind to 100 μl Ni-NTA agarose beads for 1 h while rotating gently at 4 ◦C. Unbound phages were removed by washing ten times with 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, and two subsequent washes with 1.5 ml PBS. The beads were finally resuspended in 1ml PBS. Activated and heparin-sepharose purified mast cell protease 4 was added to the resuspended beads to start the digestion of susceptible peptides. Buffer without protease was used as a control. Protease digestion was allowed to proceed at room temperature over night with gentle agitation. To recover released phages, the Ni-NTA agarose beads were pelleted by quick centrifugation and the supernatant containing the phages was removed. To ensure that all of the released phages were recovered the beads were resuspended in 100 μl PBS (pH 7.2), which was removed and added to the first supernatant after centrifugation. A control elution of the remaining phages still bound to the Ni-NTA agarose beads, using 100μl 100mM imidazole concluded that at least 1.0e8 phages were attached to the matrix during each selection round. To ensure that all phages recovered after the protease digestion step lacked the His6 tag, 15μl fresh Ni-NTA agarose beads were added to the phage suspension and the mixture agitated for 5 min followed by centrifugation to recover the supernatant. Thirty microlitres of the supernatant was used to determine the amount of detached phages in each round of selection. The remaining 1070 μl of the supernatant was added to a 10ml culture (OD~0.6) of E. coli BLT5615. The bacteria had previously been induced to produce the phage capsid protein by adding 100 μl 100m MIPTG 30 min prior to phage addition. Approximately 75 min after phage addition the bacteria lysed. The lysate was centrifuged to remove cell debris and 900 μl of the phage sub-library was added to fresh agarose beads. After binding and washing the sub-library, a new round of selection was started. Following five rounds of selection, ~100 plaques per enzyme were arbitrarily isolated from LB plates and each dissolved in phage extraction buffer (100mM NaCl and 6mM MgSO4 in 20mM Tris–HCl pH 8.0) and vigorously shaken for 30 min in order to disrupt the phages. 3412 TSVLFSLRM GTPFSLRARG PSLYSLSRV IVRYSLAGE VAMFSVRVS LANFSVRIA EVVRFSVSS LVIGYSARP ASVTLFSTR VVSLFRLGV PVVLFRLSV SPVLFRVRS PVVGQIFRVH RIATMFRVE PGGYEVLTLS RGQWPRTVA KSWYSLSSE DWSWFSLSM YFRYFSLGG WQWYSLGLH GTWFSLGVMD GGWYSVALADK MTWFSSQVA VFWRFFSNA GLFFSMYTRV VVWFRSMAD QPVFFRIRG DFQMFFRLS YSTRFFTLG GGFYTVANFRG WSFFALSRQ WNLSKFSLS SRLWRTFSLH PVLYSLGYV PGGYSVRVFNSS TLIMTFSAG GIVRFSARW GWRRVLFSAH YLTVNFSTS VRVQMFRFSHH PGGYRAMKNFAM WRTYRSAVV YMQASFALG GYQAARFAAH 44 Phage display (common panning) 5 PMID:18313755 Chymase (EC:3.4.21.39) Not determined. Not determined. Not determined. X9 T7 phage display library A 300 μl aliquot of the amplified phages (~1.0e9 pfu) was allowed to bind to 100 μl nickel-nitrilotriacetic acid (Ni-NTA) agarose beads for 1 h while rotating gently. Unbound phages were removed by washing 15 times with 1.5 ml 1M NaCl, 0.1% Tween-20 in PBS (pH 7.2), and further washed twice in 1.5 ml PBS (pH 7.2) and finally resuspended in 1ml PBS (pH 7.2). To determine the amount of phages bound to the matrix, a control elution of the phages with 100 μl 100mM imidazole was performed. This analysis showed that at least 1.0e8 phages were attached to the matrix after washing. Purified mMCP-1 (0.6 μg) was added to the tubes with the beads resuspended in PBS (pH 7.2), to start the selections. PBS (pH 7.2) without protease was used as control. The proteases were allowed to digest susceptible phages at room temperature over night under gentle agitation. To recover released phages, the Ni–NTA agarose beads were pelleted by centrifugation and the phages in the supernatant were removed in a total volume of 1000 + 100 μl PBS (pH 7.2). To ensure that all phages recovered lacked the histidine tag, 15 μl fresh Ni–NTA agarose beads were added to the phage suspension and the mixture agitated for 15 min followed by centrifugation. The supernatant was recovered and transferred to a new tube. 10 μl of the supernatant was used to determine the amount of detached phages in each round of selection. The remaining 1090 μl of the supernatant was added to a 10ml culture (OD ~0.6) of E. coli (BLT5615). The bacteria had been induced with 100 μl 100mM IPTG 30 min before phage addition, to ensure production of the phage capsid protein. Approximately 75 min after phage addition the bacteria lysed and 900 μl of the phage sublibrary was added to fresh agarose beads. After binding and washing the sub-library, a new round of selection was started. Following five rounds of selection, phages were plated on LB plates supplemented with ampicilin. Plaques corresponding to single phage clones were isolated and dissolved in phage extraction buffer (100mM NaCl, 20mM Tris–HCl pH 8.0 and 6mM MgSO4) and vigorously shaken for 30 min in order to extract the phages from the agarose. 3413 IAVFWEGTP(2) ALEVYASGG(1) AAVIAMFAGH(1) LISSYGLEL(1) SMVSYADVS(1) GQVFAEVRLQ(1) LVTRFSEEE(1) LAEYSDPAV(1) LVSVAYSET(1) GLVAYSEVE(1) GALLYTDRV(1) GILYVDSSTT(1) GVLYEESGSR(1) SVLLFEDGL(1) CDNYEDIRR(1) VASFDSAVL(1) ARRVTFDSL(1) RSTFQAALL(1) RMTFSLSTL(1) VWVVEMFTTH(1) VFDRVVFAGH(1) WTREAWALV(1) WNPQAYGLT(1) VATTFFGLT(1) HLMSFAEEW(1) LVVYGDYTT(1) GVRYFDALL(1) GGEWYEASQGG(1) WRLCPFMDA(1) AVWLYSERL(1) AWLAYTDSV(1) FLAVEAFMDH(1) WDAYNEAVE(1) GEFYSEGLGH(1) AIWWSEVVR(1) GLLYSDVGIW(1) AVSFSFTTL(1) GQAYSMALPY(1) GVLWSAFARG(1) LLLFSGGFL(1) WFRVAVYGSH(1) QVAFFGLYD(1) PGLLAFFLF(1) YVGSSVYFGH(1) LWSVYFEPM(1) FFISVYVEA(1) GFTFFDVGSY(1) [GLV]-[VAL]-[AVL]-[YF]-S-[DE]-[AVG] 47 Phage display (common panning) 5 PMID:19073880 Chymase (EC:3.4.21.39) Not determined. Not determined. Not determined. X9 T7 phage display library An aliquot of the amplified phages (~1.0e9 pfu) was allowed to bind to 100 μl Ni–NTA agarose beads for 1 h while rotating gently at 4 ◦C. Unbound phages were removed by washing 10 times with 1.5 ml 1 M NaCl, 0.1% Tween 20 in PBS, pH 7.2, and two subsequent washes with 1.5 ml PBS. The beads were finally re-suspended in 1000 μl PBS. Activated and heparin–sepharose-purified protease (~0.1 μg) was added to the re-suspended beads to start the selection of susceptible peptides. Buffer without protease was used as a control. Protease digestion was allowed to proceed at room temperature overnight with gentle agitation. To recover released phages, the Ni–NTA agarose beads were pelleted by centrifugation in a table top centrifuge and the supernatant containing the phages was removed. To ensure that all the released phages were recovered, the beads were re-suspended in 100 μl PBS (pH 7.2), and the supernatant after mixing and centrifugation was added to the first recovered phages. A control elution of the phages still bound to the beads, using 100 μl 100 mM imidazole, concluded that at least 1.0e8 phages were attached to the matrix during each selection. To ensure that the His6 tag had been hydrolyzed on all phages recovered after protease digestion, 15 μl fresh Ni–NTA agarose beads were added to the phage suspension and the mixture agitated for 15 min followed by centrifugation to recover the supernatant. Thirty microliters of the supernatant was used to determine the amount of phages detached in each round of selection. The remaining 1070 μl of the supernatant was added to a 10-ml culture (optical density ~0.6) of Escherichia coli (BLT5615). The bacteria had previously been induced to produce the T7 phage capsid protein by adding 100 μl 100 mM isopropyl β-D-1-thiogalactopyranoside 30 min prior to phage addition. The bacteria lysed ~75 min after phage addition. The lysate was centrifuged to remove ceμl debris and 900 μl of the phage sublibrary was added to 100 μl fresh agarose beads. After binding and washing the sublibrary, a new round of selection was started. Following five rounds of selection, 50 plaques were arbitrarily isolated from luria broth plates and each dissolved in phage extraction buffer (100 mM NaCl and 6 mM MgSO4 in 20 mM Tris–HCl, pH 8.0) and vigorously shaken for 30 min in order to extract the phages from the agarose. 3414 QRKLAAKLT 1 Phage display (common panning) 6 PMID:18068970 Pseudomonas aeruginosa ATCC 27853 Not determined. Not determined. Not determined. pVIII-9aa phage display library (X9) 3415 RVRSWRVRR(2) LVRPRAGRS(1) PRALRSVVR(1) PRAMRGGRR(1) PRGMRVVQR(1) TPRSARFGD(1) TLMVPRTGS(1) PRSWWKAAW(1) LVYMPRAFL(1) RSARFKSIW(1) RAAKSGLHF(1) RAFRWGRAP(1) RSRSSRLKV(1) RSSRVTSRM(1) SLRIRSSRS(1) LRLKLDIGR(1) RRQCRALRY(1) 17 Phage display (common panning) 5 PMID:22384068 Prothrombin Not determined. Not determined. Not determined. X9 T7 phage display library In brief, phages are anchored to nickel nitrilotriacetic acid (Ni-NTA) beads via the His6-tags before the first protease treatment. The protease is added and allowed to react over night, releasing phages displaying cleavage-susceptible 9-mers from the beads. Samples are centrifuged and cleaved phages are collected in the supernatant. These phages are amplified in the E. coli strain BLT5615 and enter the next selection round (biopanning). After five biopannings with 1 U of thrombin, enriched cleavage-susceptible phages are sequenced. 3416 LMPRTKRRG(2) LSPRSVRLL(1) GLMPRGVRL(1) VRARSRNSV(1) SRSKGRARN(1) RRRSSVEPF(1) PRAFKVANL(1) GGPRSWRRG(1) ARPRSWLVM(1) GFKPRTFYV(1) LVPRAYYYW(1) WGRFQGWSW(1) WTFATGPFM(1) TLMVPRTGS(1) RSVRGGRLW(1) VMRAGRWLW(1) 16 Phage display (common panning) 5 PMID:22384068 Prothrombin Not determined. Not determined. Not determined. X9 T7 phage display library In brief, phages are anchored to nickel nitrilotriacetic acid (Ni-NTA) beads via the His6-tags before the first protease treatment. The protease is added and allowed to react over night, releasing phages displaying cleavage-susceptible 9-mers from the beads. Samples are centrifuged and cleaved phages are collected in the supernatant. These phages are amplified in the E. coli strain BLT5615 and enter the next selection round (biopanning). After five biopannings with 0.2 U of thrombin, enriched cleavage-susceptible phages are sequenced. 3417 PCAIWF(58%) WVCSGG(42%) 2 Phage display (common panning) 3 PMID:27819042 Extracellular domain of vascular endothelial growth factor receptor 3 (EC:2.7.10.1) (VEGFR-3) Vascular endothelial growth factor C and D Not determined. Not determined. fUSE55-based X6 phage display library To isolate peptides targeting VEGFR-3, microtiter wells were coated with recombinant mouse VEGFR-3/Fc [1 mg in 50 ml of phosphatebuffered saline (PBS)] [10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl (pH 7.4)], blocked with PBS supplemented with 3% bovine serum albumin (BSA), and incubated with the X6 phage peptide library (e9 TU) in 50 ml of PBS supplemented with 1% BSA (PBS/BSA) for 2 hours at room temperature. The wells were then washed, and bound phages were recovered by bacterial infection and amplified for the next cycle of selection. After the third round of selection, random bacterial colonies were selected for DNA sequencing to identify the phage coding peptides. These peptides (PCAIWF and WVCSGG) specifically prevent the binding of vascular endothelial growth factor (VEGF) family members to all three receptors and downstream signaling but do not affect other angiogenic receptor tyrosine kinases (RTKs) and their ligands. One of the selected peptides (PCAIWF) was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective smallmolecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases. 3418 LPPWKLK(11/27) WSLSELH(4/27) LHRHANL(2/27) IGASVHR(1/27) AHHLKVS(1/27) NHPLYNR(1/27) ALEHTSR(1/27) HPAIRPP(1/27) SSNQFHQ(1/27) KVPGHQQ(1/27) TLAPRTA(1/27) 11 Phage display (common panning) 4 PMID:30889924 Polycrystalline SnO powder Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) For each biopanning procedure, 20 mg of the SnO powder were equilibrated for 10 min in 1 mL Tris-buffered saline + 0.1 % Tween-20 (TBST0.1) in an ultrasonic bath. After centrifugation at 10621 g for 2 min followed by removal of the supernatant, the SnO powder was resuspended in 200 μL TBST0.1. 10 μL of the phage peptide library (~1.2e11 phages) were added for 1 h under light agitation at 20 °C. To remove unbound and weakly bound phages, the SnO powder was washed 10 times with 1 mL TBST0.5. For this, first the powder was sedimented by centrifugation at 10621 g for 2 min. Subsequently, the supernatant was removed and the powder resuspended in 1 mL TBST0.5 by gentle vortexing, followed by another centrifugation step. Bound phages were eluted by 1 mL of 0.2 M Glycine-HCl (pH 2.2) supplemented with 1 mg/mL BSA. After 10 min of gentle mixing, the powder is sedimented by centrifugation, and the supernatant is transferred to a new 1.5 mL tube and neutralized by adding 150 μL of 1 M Tris–HCl (pH 9.1). The obtained M13 phages were amplified in E. coli ER2738, according to manufacturer’s recommendations. 3419 LPPWKLK(17/23) WSLSELH(2/23) LHRHANL(1/23) SSNQFHQ(1/23) VGKTHAD(1/23) FPLHELR(1/23) 6 Phage display (common panning) 5 PMID:30889924 Polycrystalline SnO powder Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) For each biopanning procedure, 20 mg of the SnO powder were equilibrated for 10 min in 1 mL Tris-buffered saline + 0.1 % Tween-20 (TBST0.1) in an ultrasonic bath. After centrifugation at 10621 g for 2 min followed by removal of the supernatant, the SnO powder was resuspended in 200 μL TBST0.1. 10 μL of the phage peptide library (~1.2e11 phages) were added for 1 h under light agitation at 20 °C. To remove unbound and weakly bound phages, the SnO powder was washed 10 times with 1 mL TBST0.5. For this, first the powder was sedimented by centrifugation at 10621 g for 2 min. Subsequently, the supernatant was removed and the powder resuspended in 1 mL TBST0.5 by gentle vortexing, followed by another centrifugation step. Bound phages were eluted by 1 mL of 0.2 M Glycine-HCl (pH 2.2) supplemented with 1 mg/mL BSA. After 10 min of gentle mixing, the powder is sedimented by centrifugation, and the supernatant is transferred to a new 1.5 mL tube and neutralized by adding 150 μL of 1 M Tris–HCl (pH 9.1). The obtained M13 phages were amplified in E. coli ER2738, according to manufacturer’s recommendations. 3420 LPPWKLK(11/27) WSLSELH(4/27) LHRHANL(2/27) IGASVHR(1/27) AHHLKVS(1/27) NHPLYNR(1/27) ALEHTSR(1/27) HPAIRPP(1/27) SSNQFHQ(1/27) KVPGHQQ(1/27) TLAPRTA(1/27) FRW 11 Phage display (in vivo) 3 PMID:30670660 Olfactory bulk Not determined. Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) To isolate peptides targeting the brain, animals received the CX8C phage library by i.v. injection. After 30 minutes in circulation, mice were perfused with 20 mL of Dulbecco’s modified Eagle’s medium (DMEM) and different regions of the brain (cerebellum, olfactory bulb and hemispheres) were collected. Phage bound to tissue were recovered by tissue homogenization followed by bacterial infection, transferred to LB media supplemented with kanamycin (100 μg/ml) and tetracylcin (20 μg/ml) amplified by overnight culture and purified from culture supernatants by the PEG/NaCl method. Two more successive rounds of selection were then performed by reinjecting i.v. the recovered pools of phage into different mice (one for each brain area). Peptides containing a conserved motif in which amino acids Phenylalanine−Arginine−Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif. 3421 CLYVNFAWRC(10) CVWANFRWQC(6) CFFADFRWYC(5) CFFVFPRWYC(2) CYSFLTSDFC(1) CFFDAIEFRC(1) CGLAVGTADC(1) CYLNWRWSVC(1) CSVNLVFGSC(1) CVYMYFGWFC(1) CMWVAWRWVC(1) CYFYNYRWWC(1) CVWMNYRWVC(1) FRW 13 Phage display (in vivo) 3 PMID:30670660 Hemisphere Not determined. Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) To isolate peptides targeting the brain, animals received the CX8C phage library by i.v. injection. After 30 minutes in circulation, mice were perfused with 20 mL of Dulbecco’s modified Eagle’s medium (DMEM) and different regions of the brain (cerebellum, olfactory bulb and hemispheres) were collected. Phage bound to tissue were recovered by tissue homogenization followed by bacterial infection, transferred to LB media supplemented with kanamycin (100 μg/ml) and tetracylcin (20 μg/ml) amplified by overnight culture and purified from culture supernatants by the PEG/NaCl method. Two more successive rounds of selection were then performed by reinjecting i.v. the recovered pools of phage into different mice (one for each brain area). Peptides containing a conserved motif in which amino acids Phenylalanine−Arginine−Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif. 3422 CIWVDFRWKC(4) CVWVDFHWVC(2) CVWVSFHWVC(2) CFWWKYVYRC(1) CVYHGFRWRC(1) CRYIDFRWSC(1) CFWYGMRWWC(1) CFWYSYRWIC(1) CRYSAWKWWC(1) CWRALNYSPC(1) CGSDGEVGRC(1) FRW 11 Phage display (in vivo) 3 PMID:30670660 Cerebellum Not determined. Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) To isolate peptides targeting the brain, animals received the CX8C phage library by i.v. injection. After 30 minutes in circulation, mice were perfused with 20 mL of Dulbecco’s modified Eagle’s medium (DMEM) and different regions of the brain (cerebellum, olfactory bulb and hemispheres) were collected. Phage bound to tissue were recovered by tissue homogenization followed by bacterial infection, transferred to LB media supplemented with kanamycin (100 μg/ml) and tetracylcin (20 μg/ml) amplified by overnight culture and purified from culture supernatants by the PEG/NaCl method. Two more successive rounds of selection were then performed by reinjecting i.v. the recovered pools of phage into different mice (one for each brain area). Peptides containing a conserved motif in which amino acids Phenylalanine−Arginine−Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif. 3423 CSWYSYRWIC CMYHNFAWYC CYYAFFRWHC CIYSFFAWRC CFFWMFRWIC CSWFDFRWHC CVYLDFKWQC CVWVSYRWVC CIYFQYHWVC CTWVNFRYVC [FYW]-X-X-[FYW]-[ARKH]-[FYW] 1021 Phage display (in vivo) 3 PMID:30670660 Brain Not determined. Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) To isolate peptides targeting the brain, animals received the CX8C phage library by i.v. injection. After 30 minutes in circulation, mice were perfused with 20 mL of Dulbecco’s modified Eagle’s medium (DMEM) and different regions of the brain (cerebellum, olfactory bulb and hemispheres) were collected. Phage bound to tissue were recovered by tissue homogenization followed by bacterial infection, transferred to LB media supplemented with kanamycin (100 μg/ml) and tetracylcin (20 μg/ml) amplified by overnight culture and purified from culture supernatants by the PEG/NaCl method. Two more successive rounds of selection were then performed by reinjecting i.v. the recovered pools of phage into different mice (one for each brain area). Peptides containing a conserved motif in which amino acids Phenylalanine−Arginine−Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif. 3424 CNARGDMHC(3) CIVRGDNVC(3) CTLRGDHHC(2) CTIRGDTHC(2) CNARGDAPC(2) CVPRGDMHC(2) CLRGDYPKC(1) CECRGDCYC(1) CRGDNTDQC(1) CVSRGDQIC(1) CVNRGDHSC(1) CNGQNWRGC(1) CLNGAYPNC(1) CSLKGDLKC(1) CSSSQSPVC(1) CHNNHYRDC(1) CHSTQMNHG(1) CFGSKAGQC(1) CHQTINRLC(1) CDRADKQGC(1) CTAATSTMC(1) CQNPNQKFC(1) RGD 22 Phage display (in vivo) 2 PMID:30730145 Blood Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Briefly, Ph.D.-C7C phage library (1.0e11 plaque forming units, or pfu) in 300 μL saline was injected into the rat through the tail vein. Forty-eight hours later, as much blood as possible was collected from the heart and plated out on LB plates containing X-gal (5-bromo-4-chloro- 3-indolyl-b-Dgalactoside) and IPTG (isopropyl-b-D-thiogalactoside). About 300 blue plaques, obtained from six rats, were picked, mixed and amplified. After purification, 1.0e11 of the amplified phage was used for second round biopanning, following the same procedure as for the first round screening but with blood withdrawn at 96 h post injection. About 500 blue plaques obtained from the second round screening were mixed, amplified, and used for the third round screening, with blood withdrawn at 120 h post injection. Thirty blue plaques each from the second and third round screening were randomly picked and subjected to DNA sequencing. The majority of the identified blood circulation-prolonging (BCP) peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1 (CNARGDMHC). The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anticancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles. 3425 CNARGDMHC(6) CIVRGDNVC(4) CVPRGDMHC(4) CLRGDYPKC(2) CECRGDCYC(2) CTIRGDTHC(2) CNARGDAPC(1) CRGDNTDQC(1) CPDATGGLC(1) CSNIMNNFC(1) CSTGWPRAC(1) CDNPDRRKC(1) CAIPNQVPC(1) CEMTNRSHC(1) CSNYMTTNC(1) CSGMYMAQC(1) 16 Phage display (in vivo) 3 PMID:30730145 Blood Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Briefly, Ph.D.-C7C phage library (1.0e11 plaque forming units, or pfu) in 300 μL saline was injected into the rat through the tail vein. Forty-eight hours later, as much blood as possible was collected from the heart and plated out on LB plates containing X-gal (5-bromo-4-chloro- 3-indolyl-b-Dgalactoside) and IPTG (isopropyl-b-D-thiogalactoside). About 300 blue plaques, obtained from six rats, were picked, mixed and amplified. After purification, 1.0e11 of the amplified phage was used for second round biopanning, following the same procedure as for the first round screening but with blood withdrawn at 96 h post injection. About 500 blue plaques obtained from the second round screening were mixed, amplified, and used for the third round screening, with blood withdrawn at 120 h post injection. Thirty blue plaques each from the second and third round screening were randomly picked and subjected to DNA sequencing. The majority of the identified blood circulation-prolonging (BCP) peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1 (CNARGDMHC). The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anticancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles.