3351 GTLMVPRTG(2) MIWAVSVGF(1) WWVAVSVHH(1) GGIAVSQWL(1) ARWAVSHHH(1) GMCAVSLFD(1) GGVVVSAVW(1) EPLLVSFWH(1) VRLLVSMHH(1) LVLLVSSLM(1) DGFLVSVFL(1) GGVYVSLTI(1) GGLFVSIRW(1) ANFFVSVAL(1) ARIMVSHHH(1) RGLMVSLHH(1) GGRQVSLFW(1) YVTFVMRTH(1) MGIFVMELL(1) GSAFVMLVP(1) WAGEVMLLR(1) GGRTVMLDF(1) ATLDVCHHH(1) VWLVVCVMH(1) AFLAVTFVH(1) SRMLVRLVF(1) EVRLVRMAH(1) YMRAVKGRH(1) IALSVKSML(1) IWSVVHHHH(1) FTLGVHHHH(1) LCTFVEAWY(1) GGLAVEVGL(1) GAMLVELHH(1) GAILVDLLG(1) RFAAVWSVW(1) FGRVVWHHH(1) GGSYVWMIT(1) TPGGVWTAC(1) WLNMVWGVL(1) TPGGVWAWL(1) GARTVWGFA(1) YWWSVWDGW(1) TPGGVFTSW(1) TPGGVFAAT(1) GGFVVFLFN(1) YLDIVYGRH(1) GGMGVYLAF(1) GREVVLRWL(1) YAEVVLLRH(1) AMEDVLHHH(1) LGRFVLHHH(1) GGEFVLWPI(1) GSLFVLYHH(1) GGWFVLAID(1) NWLYVLWDH(1) STWWVLEAH(1) GSRSVLFLC(1) TPGGVLMAW(1) WRWVVAEHH(1) LWAIVAYNW(1) YSTLVAAHH(1) GGLWVAGRW(1) GGVFVALRS(1) GFVDVALTW(1) GGSLVISLL(1) RYFEVGGGH(1) GGMWASDFC(1) GGLFASAAG(1) GGLIARYEW(1) TPGGARFAG(1) AWLDARPWR(1) WGLLANFTL(1) LWLDAGMDT(1) LDIAISLMH(1) KMELIMLHH(1) LRAIIDLGG(1) 77 Phage display (common panning) 5 PMID:30459762 Myeloblastin Not determined. Not determined. Not determined. pComb M13 phage display library An aliquot of the amplified phages were bound to Ni-NTA beads by their His6-tags. Unbound phages were removed by washing ten times in 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, with two subsequent washes with 1.5 ml PBS. The beads were finally resuspended in 1 ml PBS. PBS without protease was used as control. Phages with a random peptide that were susceptible to protease cleavage were released from the Ni-NTA matrix, and the supernatant containing these phages was recovered. To ensure that all of the released phages were recovered the beads were resuspended in 100 μl PBS (pH 7.2) and the supernatant, after mixing and centrifugation, was added to the first supernatant. To ensure that the His6-tags had been hydrolyzed on all phages recovered after protease digestion, 15 μl fresh Ni-NTA agarose beads were added to the combined phage supernatant and the mixture agitated for 15 min followed by centrifugation. A control elution of the phages still bound to the beads, using 100 μl 100 mM imidazole showed that at least 1 × 10^8 phages were attached to the matrix during each selection. 3352 GWAVFSDIV(2) GVCSFWESG(2) TALLWLELT(2) GVIMLLETI(2) PFALWQECH(1) GGFLFSDAL(1) LTFVFSDHH(1) GVTVFSDSH(1) TLSPFMEML(1) GGSWFMEWE(1) LDWFFYEIV(1) GLTTFVDGA(1) GGAVFVDLG(1) RNAAFLDQH(1) EASIFLESH(1) GFTLFLEDR(1) GMDLFLDTW(1) GGTEFLELV(1) GMTPFADVC(1) GGFVFADHV(1) GMLLWEEWA(1) GGELFRELY(1) WVTLFHEPH(1) FLILLSEEH(1) GGVPLTELL(1) GGRRLSDDV(1) GGVVLMDSV(1) VCQLLMDHH(1) DLYLLVDDH(1) VYLQLVDEH(1) GQMTLVDLH(1) YFVILLDHH(1) GVVVLLDNL(1) AFLLLIEAF(1) VEVLLLEWV(1) YVPLLLDFW(1) GGWFLLDWE(1) WEMLLWDHH(1) RAFVLWDTH(1) GGECLYDAA(1) RVLTLFDWH(1) GGITLFELV(1) VGTGLVRMN(1) RRAELPGGL(1) VVTFFAMDH(1) GGGYYLMLD(1) MLDFFRFHH(1) GVWWYKTWC(1) ELMVYRSHH(1) PEILWRMVH(1) GRRGWRRHH(1) GGGAWRLML(1) GDCEYEGGK(1) GVTLFVWAR(1) GGELFAQCL(1) GLELFAFAV(1) GGLEWLWGC(1) LLRVWVLSV(1) GGLKWLFTR(1) GCFMWSGSW(1) GGGCFSVWS(1) GLVSFSVYV(1) GGTVFTSLL(1) GTEAFGGGG(1) NVLCFVLGV(1) GATSYVGCV(1) GGGMWGATT(1) AEGLWGSVR(1) GKGLWGPRR(1) 69 Phage display (common panning) 5 PMID:30459762 Cathepsin G (EC:3.4.21.20) Not determined. Not determined. Not determined. CX10C T7 phage display library An aliquot of the amplified phages were bound to Ni-NTA beads by their His6-tags. Unbound phages were removed by washing ten times in 1.5 ml 1 M NaCl, 0.1% Tween-20 in PBS, pH 7.2, with two subsequent washes with 1.5 ml PBS. The beads were finally resuspended in 1 ml PBS. PBS without protease was used as control. Phages with a random peptide that were susceptible to protease cleavage were released from the Ni-NTA matrix, and the supernatant containing these phages was recovered. To ensure that all of the released phages were recovered the beads were resuspended in 100 μl PBS (pH 7.2) and the supernatant, after mixing and centrifugation, was added to the first supernatant. To ensure that the His6-tags had been hydrolyzed on all phages recovered after protease digestion, 15 μl fresh Ni-NTA agarose beads were added to the combined phage supernatant and the mixture agitated for 15 min followed by centrifugation. A control elution of the phages still bound to the beads, using 100 μl 100 mM imidazole showed that at least 1 × 10^8 phages were attached to the matrix during each selection. 3353 HWWWPAS(6)[0.60] HWTWWNL(1)[0.58] QNTLHPR(1)[0.06] VRIPVWH(1)[0.09] HWWW 4 Phage display (competitive panning) 3 PMID: Lysozyme Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The plate was read at 405 nm using a model M550 microplate reader (BIO-RAD, US). The library phages served as the negative control. ELISA signal values were reprodcued from Figure 4 and shown. The signal of library phages were 0.07. In elution step, the wells were eluted four times and individual elution samples were collected for further assays and/or phage amplification. The elution in each round was conducted as follows: (1) 0.1M glycine–HCl buffer (pH 2.2) containing 1 mg/ml BSA was added in the wells and incubated for 10 min; (2) 200 μl of 0.2 mg/ml lysozyme solution was added and incubated for 60 min; (3) the glycine–HCl buffer was added in the wells again, incubating for 10 min; and (4) the final elution was done by adding 200 μl of 0.2 mg/ml lysozyme solution in the wells and incubating for 120 min. Through comparison of the DNA sequences of foreign peptides of the clones showing specificity to lysozyme molecules, the HWWW motif was found to be the necessary amino acid sequence for the affinity. The electrostatic and hydrophobic interactions are considered to contribute to the affinity for the protein. Moreover, protein chromatography with the immobilized HWWWPAS on Sepharose gel indicated the strong binding affinity of the peptide for lysozyme. 3354 HWWWPAS(15)[1.00] 1 Phage display (competitive panning) 3 PMID: Isulin Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The ELISA signal of the original library phages was used as negative control. It was found that the ELISA signals of the collected phages for insulin reached over 1.0, while that of the control was only 0.164. For the elution of bound phages, the wells were incubated with 0.1 M glycine-HCl buffer (pH 2.2) for 10 min and then with 200 μl of 0.2 mg/mL insulin solution for 60 min. These two elution steps were repeated and the phages collected in the fourth elution were amplified for the next round biopanning and screening. The peptide (HWWWPAS) was synthesized and coupled to EAH Sepharose 4B (5.4 μmol/mL bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6 mg/mL bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin. 3355 GGVCFRVGLSIVCNF ALVFFFSSGDFYGVS GSCRAFLSGVVCSFP 3 Phage display (common panning) 4 PMID:15377604 Lactotransferrin (EC:3.4.21.-), Lactoferrin Not determined. Not determined. Not determined. f3-15mer phage display library (X15) The custom synthesized peptide HuH6 (GSCRAFLSGVVCSFP) displayed on the HuLF binding phage isolated from the linear pentadecamer library, however, did not bind lactoferrin, as detected with Surface Plasmon Resonance, nor did it inhibit the phage binding to lactoferrin. 3356 CAPRQPPC CRTSLKVC CDTNREQC CDQDQDTC CDQVAKGC CEKGQRRC CEGKQRRC CTKGNQPC CTRSGTRC CSATEHGC CPGGYTKC CTDNAKEC CDVGQNKC CHQHRQRC 14 Phage display (common panning) 2 PMID:15377604 Lactotransferrin (EC:3.4.21.-), Lactoferrin Not determined. Not determined. Not determined. CX6C phage display library The peptides HuN (CEGKQRRC) and HuL (CDQVAKGC) selected from the cyclic hexamer library, custom synthesized did neither bind lactoferrin nor inhibit the phage binding to lactoferrin. Furthermore, reducing both peptides to obtain a linear form did not change this. 3357 CIPASKAC CGLTGLAC CDRPNAKC CPKHRNKC CPRSQQEC CQWLQHPC CPYTYWTC CTAPRRAC CRKSVHSC CSKVKNRC CSFRGQSC CYQSQNGC CHGKSANC CGKFSYQC CPKTERRC 15 Phage display (common panning) 2 PMID:15377604 Bovine lactoferrin Not determined. Not determined. Not determined. CX6C phage display library 3358 TRWLVYFSRPYLVAT(8)[4.4] PRHVFYRWFLSNPRI(4)[NT] IVRHLFLHVYPRVLM(1)[NT] 3 Phage display (common panning) 4 PMID: Barley α-amylase 1 Not determined. Not determined. Not determined. f3-15mer phage display library (X15) ELISA The phage clone was analyzed by measuring the dissociation constant using an indirect competition method based on ELISA. All three peptides share a common feature of an Arg residue flanked by high numbers of Tyr and Trp/Phe residues. The phage display peptide TRWLVYFSRPYLVAT showed an activity 5-fold greater than those of the other two peptides and a dissociation constant of 4.4 nM. 3359 RLRSFY HLNSLS RVLSAH KGTTYY EINVPT SFAKLS LPPPKL SHLPWH 8 Phage display (common panning) 3 PMID:8804536 von Willebrand factor, vWF Type I collagen Not determined. Not determined. f3-6mer phage display library (X6) Phages from the 6-mer bacteriophage display library were added to von Willebrand Factor (vWF). The solution was then transferred into the recombinant Factor VIII (rFVIII) coated microtiter wells to effect capture of vWF and vWF:phage complexes by recombinant Factor VIII, which was designed to avoid selection of peptides binding to vWF at FVIII contact sites. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCI at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9e6 to 2e6 (1/M). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK. 3360 KHRFNKD(2)[1.62 ± 0.23] PIISRAT(2)[0.15 ± 0.02] WSMRGTT(1)[NT] PGPSTTS(1)[NT] SKPLHKM(1)[NT] HRRPSRS(1)[NT] QISSLRA(1)[NT] WTDPHPP(1)[NT] HLAPAQY(1)[NT] 9 Phage display (competitive panning) 4 PMID: Anti-goat immunoglobulin G monoclonal antibody Goat immunoglobulin G Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The immunoplate and absorbance was estimated at 405 nm using a Synergy-HT microplate reader (Biotek, Winooski, USA). Each value was obtained by subtracting the value for the control from the absorbance value corresponding to each peptide. The control absorbance was determined using an immunoplate treated with blocking buffer only. The absorbance value was reproduced from Figure 2 and shown. Goat immunoglobulin G (IgG, 150 μg) was added to a culture dish with 1.5 mL TBST (0.1% Tween-20, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5). The dish was incubated overnight at 4℃ and washed with TBST. Then, the dish was filled with blocking buffer (1 mM EDTA, 0.25% BSA in TBST), incubated at room temperature for 1 h, and washed with TBST. Rabbit anti-goat IgG antibody (27 μg) was added to the dish with 1 mL TBS (150 mM NaCl, 50 mM Tris-Cl, pH 7.5) and allowed to react at room temperature for 1 h. After washing, phages (2e11 pfu diluted in 1 mL TBS) were added to the dish and allowed to react at room temperature for 1 h. Rabbit anti-goat IgG antibody (27 μg in 1 mL TBS) was added to the dish and allowed to react at room temperature for 1 h for competitive elution of phages specific to rabbit antibody. One of these sequences (KHRFNKD), which was rich in positive charge and homologous to the IgG-binding domains of Staphylococcal protein A, exhibited a high and specific affinity to rabbit antibody. This peptide was immobilized on a quartz crystal microbalance (QCM) biosensor, and the resulting QCM was found to be capable of capturing antibody and detecting antigen, with potential applications in antibody purification and enzyme immunoassay. 3361 CPLSPSHEC(5)[0.08 ± 0.03] CAAPSPRNC(1)[NT] CPRTEKRRC(1)[NT] CQRSGKPSC(1)[NT] CPAGADWPC(1)[NT] CEPTSHFHC(1)[NT] CSSGSDYTC(1)[NT] 7 Phage display (competitive panning) 4 PMID: Anti-goat immunoglobulin G monoclonal antibody Goat immunoglobulin G Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA The immunoplate and absorbance was estimated at 405 nm using a Synergy-HT microplate reader (Biotek, Winooski, USA). Each value was obtained by subtracting the value for the control from the absorbance value corresponding to each peptide. The control absorbance was determined using an immunoplate treated with blocking buffer only. The absorbance value was reproduced from Figure 2 and shown. Goat immunoglobulin G (IgG, 150 μg) was added to a culture dish with 1.5 mL TBST (0.1% Tween-20, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5). The dish was incubated overnight at 4℃ and washed with TBST. Then, the dish was filled with blocking buffer (1 mM EDTA, 0.25% BSA in TBST), incubated at room temperature for 1 h, and washed with TBST. Rabbit anti-goat IgG antibody (27 μg) was added to the dish with 1 mL TBS (150 mM NaCl, 50 mM Tris-Cl, pH 7.5) and allowed to react at room temperature for 1 h. After washing, phages (2e11 pfu diluted in 1 mL TBS) were added to the dish and allowed to react at room temperature for 1 h. Rabbit anti-goat IgG antibody (27 μg in 1 mL TBS) was added to the dish and allowed to react at room temperature for 1 h for competitive elution of phages specific to rabbit antibody. 3362 SAGHLTHGGGWAVPFYSHSTPARYAN YFPKWKVGGGSAVPFYSHSMESASAL SPLIWNWGGGSAVPFYSHSHMKSQSP DVNKYYLGGGSAVPFYSHSSRSEDYN HPFLRLPGGGSAVPFYSHSFQSPMLP 5 Phage display (competitive panning) 3 PMID:11397457 Lipase (EC:3.1.1.3) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Biopanning was performed in microtiter plate wells. Elution of the phage was carried out using 100 μl of 100 μg/ml lipase solution in TBST. 3363 HYSPFRTGGGSAVPFYSHSPSTFMSR 1 Phage display (competitive panning) 6 PMID:11397457 Lipase (EC:3.1.1.3) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Biopanning was performed in microtiter plate wells. Elution of the phage was carried out using 100 μl of 100 μg/ml lipase solution in TBST. 3364 SSRLMIL AAWLSPL GSFLYHP SFTHLLY LTPVYYW SSHMPHW 6 Phage display (competitive panning) 3 PMID:11397457 Lipase (EC:3.1.1.3) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Biopanning was performed against lipase attached to magnetic beads. Bound phage were eluted by resuspending the beads in 100 μl of 100 μg/ml lipase solution in TBST, incubated at room temperature for 1 h. The synthetic heptapeptide GSFLYHP did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. 3365 GETRAPL(3)[n.a.] TVRTSAD(2)[n.a.] YPSPWGY(2)[n.a.] ISLPSPT(1)[0.25 ± 0.02] SSQLSRP(1)[0.24 ± 0.01] YNSPTHH(1)[0.30 ± 0.03] APKQSLE(1)[n.a.] APTAVSK(1)[n.a.] ETTHLTG(1)[n.a.] GGLSSRP(1)[n.a.] GVPTALP(1)[n.a.] KPFPPMK(1)[n.a.] LGTSMQL(1)[n.a.] LPLWEVY(1)[n.a.] LTAQPST(1)[n.a.] MISSNSS(1)[n.a.] QLKPLEF(1)[n.a.] QLVYPAP(1)[n.a.] SGTHHKA(1)[n.a.] SLGPSPG(1)[n.a.] SPHDVAYD(1)[n.a.] TLNRVPN(1)[n.a.] TNTMTRA(1)[n.a.] TPHPLRL(1)[n.a.] VNRSSLY(1)[n.a.] VSTPSTP(1)[n.a.] 26 Phage display (common panning) 4 PMID:30268850 Cystic fibrosis (CF) mucus Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA To understand binding affinities between phage-displayed peptides and the major protein component of mucus (mucin), ELISA was performed. A mucin solution was used instead of CF mucus since other mucus components such as lecithin or BSA might block the binding of mucin protein to the wells. Phage clones SSQ-phage, ISL-phage, and YNS-phage binding to mucin was measured by ELISA and compared to wild-type phage. The anti-M13 antibody used for detection binds specifically to an epitope on the pVIII major coat protein. Thus the binding affinities measured correlate with phage-mucin interactions. M13KE wild-type phage demonstrated a statistically significant higher binding affinity for mucin than phage clones SSQ-phage, ISL-phage, and YNS-phage Mucin only and monoclonal antibody only controls did not show specific binding, confirming that the measured binding affinities are specific to interactions between phage and mucin. The affinity value was measured at 450nm. The absorbance (450nm) value was reproduced from Figure 5 and shown. An initial concentration of 2e10 phage pfu was mixed with cystic fibrosis (CF) mucus, and 250 μL of the mixture was transferred to the apical compartment (donor) in the transwell system containing 1.5 mL phosphate buffered saline (PBS) in the basolateral compartment (receiver) and incubated for 1 h at room temperature (25°C). After 1 h, the eluate was collected from the basolateral side and titered using standard double-layer plaque assay to quantify phage concentration. The eluted phage library was amplified in XL-1 Blue E. coli (Agilent, CA) to make more copies, which were quantified by plaque assay prior to the next round of screening. Equivalent amounts of input phage were added for each round. This iterative screening was performed for a total of four rounds. Here, phage clones displaying discovered peptides were shown to have up to 2.6-fold enhanced diffusivity in the CF mucus model. In addition, we demonstrate reduced binding affinities to mucin compared to wild-type control. 3366 GCTRMICN(2) GCTRMLCR(2) GCTRMMCR(2) ACTKMMCQ(1) ACTRMACA(1) ACTRMRCS(1) GCTKKMCG(1) GCTKMICR(1) GCTKMLCG(1) GCTKMLCL(1) GCTKMMCG(1) GCTKQLCV(1) GCTRIMCG(1) GCTRMGCV(1) GCTRMICG(1) GCTRMLCG(1) GCTRMLCN(1) GCTRMLCT(1) GCTRMLCS(1) GCTRMMCA(1) GCTRMMCE(1) GCTRMMCS(1) GCTRMQCS(1) GCTRMVCG(1) GCTRMVCM(1) GCSRMICE(1) GCSRMLCD(1) GCSRMLCG(1) GCSRMLCV(1) GCSRMMCG(1) GCSRMRCD(1) LCTKMYCG(1) MCTKMLCK(1) MCTRMFCM(1) MCTRMMCR(1) MCTRMSCF(1) NCTRMMCN(1) QCTRMFCS(1) SCTRMACQ(1) SCTRMACR(1) SCTRMHCG(1) SCTRMLCG(1) SCTRMRCN(1) SCTRMSCG(1) SCSRMLCF(1) YCTRMMCE(1) 46 Phage display (common panning) 3 PMID:30543823 Trypsin Pancreatic trypsin inhibitor Not determined. Not determined. SGPI-2 XCX4CX M13 phage display library Briefly, bovine trypsin was immobilized on MaxiSorp (Nunc) plates. Phage particles were obtained with the PEG/NaCl precipitation method and applied to the blocked wells. Following thorough washing, bound phages were eluted. E. coli XL1-Blue was used to amplify the eluted phages to be used in a next round of selection. After three selection and amplification cycles, the libraries were enriched over 100-fold compared to the control obtained on blocked wells containing no target enzymes. 3367 GCARHFAP(1) GCARIYSP(1) GCARNFDP(1) GCDKASRF(1) GCDRRLSF(1) GCDRSLNY(1) GCDRSVMY(1) GCIKLYSP(1) GCNKGLWM(1) GCNKIVHP(1) GCNRILDW(1) GCPRFLDF(1) GCPRGLSF(1) GCPRHLVP(1) GCPRHYIP(1) GCPRHYYP(1) GCPRIIYP(1) GCPRILAS(1) GCPRILDY(1) GCPRILQP(1) GCPRILSL(1) GCPRIMAP(1) GCPRIYFP(1) GCPRLFEF(1) GCPRLFHF(1) GCPRLLSP(1) GCPRLYLP(1) GCPRMVDA(1) GCPRSLDF(1) GCPRVHFP(1) GCPRVINP(1) GCPRVLDT(1) GCPRVVDL(1) GCPRYLSL(1) GCRRNIDP(1) GCTKIYDP(1) GCTRDLRL(1) GCTRELAP(1) GCTRIFSP(1) GCTRSLDF(1) GCSKQLSF(1) GCSRALNF(1) GCSRHLHY(1) GCSRHLNP(1) GCSRHLSP(1) GCSRIFNP(1) GCSRIHAP(1) GCSRILNP(1) GCSRMLDP(1) GCSRNFEP(1) GCSRNYAP(1) GCSRQLDL(1) GCSRVLSF(1) GCSRVRDD(1) GCSRVYAP(1) GCSRVYSP(1) 56 Phage display (common panning) 3 PMID:30543823 Trypsin Pancreatic trypsin inhibitor Not determined. Not determined. SPINK1 GCX6 M13 phage display library Briefly, bovine trypsin was immobilized on MaxiSorp (Nunc) plates. Phage particles were obtained with the PEG/NaCl precipitation method and applied to the blocked wells. Following thorough washing, bound phages were eluted. E. coli XL1-Blue was used to amplify the eluted phages to be used in a next round of selection. After three selection and amplification cycles, the libraries were enriched over 100-fold compared to the control obtained on blocked wells containing no target enzymes. 3368 ACTLKMCK(1) ACTLRACI(1) ACTMKMCR(1) ACTMMMCK(1) ACTMRACP(1) ACTYRRCA(1) DCTFKLCR(1) DCTMKFCQ(1) DCTMRRCK(1) DCTQKACR(1) DCTWKLCR(1) DCTWKRCT(1) DCTWRLCN(1) DCTWRMCR(1) DCTWRRCR(1) GCSWMRCS(1) GCTFMRCQ(1) GCTLKMCR(1) GCTLRLCA(1) GCTLRRCR(1) GCTMIRCK(1) GCTMKLCG(1) GCTMKLCR(1) GCTMKRCK(1) GCTMMLCR(1) GCTMMMCK(1) GCTMRACR(1) GCTMRMCQ(1) GCTMRRCR(1) GCTMVMCR(1) GCTMVRCK(1) GCTWKKCR(1) LCTYRMCR(1) MCTMMWCR(1) MCTWKMCA(1) MCTWKMCK(1) SCSWKRCN(1) SCSWKRCR(1) SCTLKLCM(1) SCTLKRCK(1) SCTMKLCK(1) SCTMKMCK(1) SCTMKRCR(1) SCTMRRCK(1) SCTMRRCR(1) SCTWRLCK(1) TCTLKRCR(1) TCTMKMCR(1) VCTMKMCR(1) VCTWKMCG(1) VCTWKMCR(1) VCTYALCR(1) 52 Phage display (common panning) 3 PMID:30543823 Chymotrypsin Not determined. Not determined. Not determined. SGPI-2 XCX4CX M13 phage display library Briefly, chymotrypsin was immobilized on MaxiSorp (Nunc) plates. Phage particles were obtained with the PEG/NaCl precipitation method and applied to the blocked wells. Following thorough washing, bound phages were eluted. E. coli XL1-Blue was used to amplify the eluted phages to be used in a next round of selection. After three selection and amplification cycles, the libraries were enriched over 100-fold compared to the control obtained on blocked wells containing no target enzymes. 3369 GCAFVMKP(1) GCPYMIRP(1) GCAYVLRP(1) GCTLVFRP(1) GCAFELRP(1) GCSFVLKP(1) GCNFKLSP(1) GCTLMYRP(1) GCPLILKP(1) GCSFIMRP(1) GCAFTLRP(1) GCPYKLSW(1) GCPFLLRF(1) GCTYLHRP(1) GCSWIYKP(1) GCTWLYRP(1) GCPFALRL(1) GCSFQLRQ(1) GCLFQLRP(1) GCSFELRP(1) GCSWVLSP(1) GCSFILRP(1) GCTYLYRP(1) GCTFVLKP(1) GCNFMFRP(1) GCAYLLRP(1) GCHFILKP(1) GCTFILRP(1) GCTFVYSP(1) GCSWQLRP(1) GCTFLLRP(1) GCVLSLRP(1) GCTFYLKP(1) GCVYIFRP(1) GCTMILKP(1) GCTLQLRP(1) GCAMILRP(1) GCGWILRP(1) GCSWVLRP(1) GCAYTLRP(1) GCTMVLRP(1) GCNYILKP(1) GCQWSLRP(1) GCTMEWRP(1) GCSMIFKP(1) GCTFVLRP(1) GCFYLLRP(1) GCPFILKL(1) GCPLIFKP(1) GCTYILRP(1) GCSFLLRP(1) GCPYVLKP(1) GCALMLRP(1) GCAFVIRP(1) GCTFSLRA(1) GCAFSLRR(1) 56 Phage display (common panning) 3 PMID:30543823 Chymotrypsin Not determined. Not determined. Not determined. SPINK1 GCX6 M13 phage display library Briefly, chymotrypsin was immobilized on MaxiSorp (Nunc) plates. Phage particles were obtained with the PEG/NaCl precipitation method and applied to the blocked wells. Following thorough washing, bound phages were eluted. E. coli XL1-Blue was used to amplify the eluted phages to be used in a next round of selection. After three selection and amplification cycles, the libraries were enriched over 100-fold compared to the control obtained on blocked wells containing no target enzymes. 3370 KKQKCRTDACVTQM(7)[128.6 ± 23.5] NEKKCKGARCTTVT(3)[84.3 ± 19.4] ATPKCKKKSCMTTQ(3)[94.5 ± 29.9] VDKKCKSDDCGAWH(3)[n.a.] ETKKCTTGPCKVVT(2)[13.4 ± 4.7] HDKKCKRQPCVLAN(2)[n.a.] FDKKCKSNKCLEVR(2)[n.a.] PKKKCHPEPCQTCG(2)[n.a.] KTEHCKKRKCPLDM(2)[n.a.] KKKKCKKKICTTHT(1)[n.a.] KKKKCKKNTCKNHT(1)[n.a.] 11 Phage display (common panning) 3 PMID:29928986 Terbium doped cerium-magnesium aluminate (CAT) Not determined. Not determined. Not determined. F88-FUSE X4CX4CX4 phage display library (X4CX4CX4) Binding assay Pre-identified phage, derived from single clone amplification out of colonies of infected cells, were characterized concerning their ability to bind to individual components of fluorescent lamp powder and to quantify the selective binding behavior. Components tested were CeMgAl11O19:Tb3þ (CAT), Y2O3:Eu3þ (YOX), BaMgAl10O17:Eu2þ (BAM), LaPO4:Ce3þ,Tb3þ (LAP) and halophosphate (HP). Mineral preparation, phage production and binding studies were performed. Mineral dispersions were incubated together with the phage and subsequently repeatedly washed, resulting in the removal of weakly bound phage. Phage concentrations in input and eluate were determined and compared to the binding behavior of wild-type fd-tet phage (obtained by Dr. Jamie Scott). Binding efficiency was calculated using phage titer before and after binding assays in comparison to the wild-type phage. Random peptide sequences with high affinity to CAT minerals were identified from the PVIII phage library Cys4. After three rounds of biopanning corresponding DNA sequences of the displayed peptides on phage of 80 infected colonies were amplified via PCR and sequenced. 11 different sequences were determined. 3371 CMSTGLSSC(28)[12.8 ± 0.7] CVPILEGTC(5)[21.5 ± 0.3] CDRTISNKC(2)[31.8 ± 0.5] CQNPGNTLC(2)[2.5 ± 0.9] CSGTGASYC(2)[1.7 ± 0.7] CSSSVVTHC(2)[2.3 ± 0.9] CDAKDLNSC(1)[2.5 ± 0.9] CDNDTKASC(1)[1.5 ± 0.5] CGLTDTSNC(1)[0.8 ± 0.3] CKTSTHAIC(1)[1.5 ± 0.3] CMRDSKMLC(1)[2.2 ± 0.6] CSTISKAKC(1)[2.6 ± 0.8] CTASQNFYC(1)[1.4 ± 0.2] CTGQGGEYC(1)[1.5 ± 0.2] CTKTQTHAC(1)[0.8 ± 0.0] CTNHSAYHC(1)[2.7 ± 2.0] CTQMLGQLC(1)[1.4 ± 0.27] CVSPNKEAC(1)[3.1 ± 0.7] 18 Phage display (common panning) 3 PMID:29928986 Serum 13006 from patients sensitized to soy bean Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Binding assay Pre-identified phage, derived from single clone amplification out of colonies of infected cells, were characterized concerning their ability to bind to individual components of fluorescent lamp powder and to quantify the selective binding behavior. Components tested were CeMgAl11O19:Tb3þ (CAT), Y2O3:Eu3þ (YOX), BaMgAl10O17:Eu2þ (BAM), LaPO4:Ce3þ,Tb3þ (LAP) and halophosphate (HP). Mineral preparation, phage production and binding studies were performed. Mineral dispersions were incubated together with the phage and subsequently repeatedly washed, resulting in the removal of weakly bound phage. Phage concentrations in input and eluate were determined and compared to the binding behavior of wild-type fd-tet phage (obtained by Dr. Jamie Scott). Binding efficiency was calculated using phage titer before and after binding assays in comparison to the wild-type phage. Biopanning was carried out on planar sol–gel surfaces. Sol-gels with cation exchange properties were fabricated using Dispex N40, polyacrylic acid or polystyrene sulfonate.63 colonies were examined for their peptide sequence. 3372 CSGTGASYC(3)[5.9 ± 1.7] CMSTGLSSC(2)[4.0 ± 2.9] CNTGSPYEC(2)[1.2 ± 0.0] CTASQNFYC(2)[10.4 ± 0.0] CGSRSAQTC(1)[2.0 ± 1.1] CGTKGSLNC(1)[17.8 ± 3.0] CGYSSFNRC(1)[3.6 ± 1.2] CHHPVANTC(1)[1.1 ± 0.2] CHNETQKMC(1)[1.6 ± 0.6] CKDTSRSAC(1)[1.2 ± 0.1] CNAKHHPRC(1)[16.8 ± 6.5] CNGRAVNYC(1)[0.9 ± 0.5] CPGASVTYC(1)[1.1 ± 1.0] CRAEGTSEC(1)[1.0 ± 0.0] CRGATPMSC(1)[1.0 ± 0.7] CSLATDQKC(1)[2.1 ± 0.8] CSNNHSSMC(1)[2.2 ± 1.0] CSTATPYKC(1)[2.4 ± 1.5] CTKTDVHFC(1)[2.6 ± 2.0] CTSVLNNTC(1)[2.4 ± 0.4] CVPILEGTC(1)[1.9 ± 0.8] 21 Phage display (common panning) 3 PMID:29928986 Nickel ion Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Binding assay Pre-identified phage, derived from single clone amplification out of colonies of infected cells, were characterized concerning their ability to bind to individual components of fluorescent lamp powder and to quantify the selective binding behavior. Components tested were CeMgAl11O19:Tb3þ (CAT), Y2O3:Eu3þ (YOX), BaMgAl10O17:Eu2þ (BAM), LaPO4:Ce3þ,Tb3þ (LAP) and halophosphate (HP). Mineral preparation, phage production and binding studies were performed. Mineral dispersions were incubated together with the phage and subsequently repeatedly washed, resulting in the removal of weakly bound phage. Phage concentrations in input and eluate were determined and compared to the binding behavior of wild-type fd-tet phage (obtained by Dr. Jamie Scott). Binding efficiency was calculated using phage titer before and after binding assays in comparison to the wild-type phage. Biopanning was carried out on planar sol–gel surfaces. Sol-gels with cation exchange properties were fabricated using Dispex N40, polyacrylic acid or polystyrene sulfonate.29 colonies were examined for their peptide sequence. 3373 KVWILPA(6/30) KVWVIPS(4/30) 2 Phage display (common panning) 3 PMID:30521966 Virus-like particle (VLP) of orange-spotted grouper nervous necrosis virus (OGNNV) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Briefly, 150 μL of the purified RBS (100 μg/mL) were coated with coating buffer (0.1 M NaHCO3, pH 8.6) in each well of the 96-well plate. After blocking with BSA (5 mg/mL) in coating buffer and washing 5 times with 0.1% TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% [v/v] Tween-20, pH 7.5), the well containing RBS was incubated with 100 μL of diluted library (5 × 1011 sequences) for 1 h at room temperature. After washed 10 times with TBST, bound phages were eluted with elution buffer (0.2 M glycine, 1 mg/mL BSA, pH 2.2) and neutralized with 1 M Tris (pH 9.1). Eluates were incubated with Escherichia coli ER2738 cells and amplified for 5 h at 37 °C. The amplified phages were collected, purified, and titrated for the next round of surface panning procedure. 3374 VHWDFRQWWQPS(11/30) 1 Phage display (common panning) 3 PMID:30521966 Virus-like particle (VLP) of orange-spotted grouper nervous necrosis virus (OGNNV) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) Briefly, 150 μL of the purified RBS (100 μg/mL) were coated with coating buffer (0.1 M NaHCO3, pH 8.6) in each well of the 96-well plate. After blocking with BSA (5 mg/mL) in coating buffer and washing 5 times with 0.1% TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% [v/v] Tween-20, pH 7.5), the well containing RBS was incubated with 100 μL of diluted library (5 × 1011 sequences) for 1 h at room temperature. After washed 10 times with TBST, bound phages were eluted with elution buffer (0.2 M glycine, 1 mg/mL BSA, pH 2.2) and neutralized with 1 M Tris (pH 9.1). Eluates were incubated with Escherichia coli ER2738 cells and amplified for 5 h at 37 °C. The amplified phages were collected, purified, and titrated for the next round of surface panning procedure. From the positive clones, a dodecapeptide (VHWDFRQWWQPS) with the highest binding capacity (BC) to RBS (virus-like particle (VLP) of orange-spotted grouper nervous necrosis virus (OGNNV)) was selected. This affinity peptide (AFP) agglutinated or disrupted virion particles, inhibiting RBS entry into sea bass (SB) cells. To enhance BC and solubility, we amended the AFP sequence as “LHWDFQSWVPLL” and named as 12C. One to three copies of 12C in tandem were prokaryotically expressed with a maltose binding protein (MBP) linked by a flexible peptide. Of the recombinant proteins expressed, MBP-triple-12C (MBP-T12C) exhibited the highest BC, efficiently blocked RBS entry, and strongly inhibited OGNNV infection at viral entry. Moreover, MBP-T12C bound the VLPs of all nervous necrosis virus (NNV) serotypes, displaying broad-spectrum anti-NNV ability, and recognized only OGNNV and mud crab virus, demonstrating binding specificity. Therefore, these anti-NNV AFPs specifically bound NNV, aggregating or disrupting the viral particles, to reduce the contact probability between the virus and cell surface, subsequently inhibiting viral infection. Our results not only provided a candidate of anti-NNV AFP, but a framework for the development of antiviral AFP. 3375 CDNVAQSVC(6)[4059.0 ± 213.5] CKTSPWAKC(1)[n.a.] 2 Phage display (common panning) 3 PMID:30431079 C57BL/6 mouse bone marrow mesenchymal stem cell (BMMSC) Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) Flow Cytometry The identified peptide (CDNVAQSVC) was designated as a mouse BMMSCs affinity peptide (D7). A peptide (CVAVQNDSC) with the same amino acids as D7 in a scrambled order was used as the negative control and termed V7. RGD, a peptide composed of 3 amino acids (arginine, glycine and aspartic acid), was used as the positive control. The C57BL/6 mouse BMMSCs were incubated for 1 h with FITC-labeled D7, V7 or RGD, and measured via flow cytometry. The average fluorescence intensity was 3,058.5 ± 604.6 for the mouse BMMSCs incubated with FITC-RGD, and 146.5 ± 60.1 for the mouse BMMSCs incubated with FITC-V7. The average fluorescence intensity was shown for the mouse BMMSCs incubated with FITC-D7. The phage display library consisted of e9 different phages. Cells plated in 60x15 mm petri dishes were rinsed twice with PBS (Biological Industries, Ltd.; cat. no., 02-024-1A) and pre-blocked with DMEM containing 0.5% BSA (albumin bovine fraction-V; NeoFroxx, Einhausen, Germany; cat. no., GRM105-5G) without supplements at 37˚C and 5% CO2 for 1 h. The Ph.D.C7C™ Phage Display Library (1e11 PFU) was then introduced to the cells. Non-binding phages were then discarded, and the cells were washed 5 times in cold PBS. The phages binding to the cells were eluted with 1 ml Glycine/HCl (pH 2.2) with 1 mg/ml BSA for 20 min at room temperature while being gently shaken. The eluted phages were collected and neutralized with 0.15 ml 1 M Tris-HCl (pH 9.1). A total of 3 rounds of panning were performed for each sample. A peptide sequence (CDNVAQSVC), termed D7, was identified through phage display technology using C57BL/6 mouse BMMSCs. Subsequent analysis suggested that the identified loop-constrained heptapeptide exhibited a high specific affinity for mouse BMMSCs. Due to this specific affinity for BMMSCs, the present study provides a selective method to improve mesenchymal stem cell (MSC) based tissue engineering (TE) strategies for the treatment of osteonecrosis of the femoral head (ONFH).