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Ph d 7 phage display peptide library kit

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The Ph.D.-7 Phage Display Peptide Library Kit is a tool for generating and screening peptide libraries. The kit includes a phage display library containing approximately 10^9 random 7-mer peptide sequences displayed on the surface of M13 phage. The library can be used to identify peptides that bind to a target of interest.

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9 protocols using ph d 7 phage display peptide library kit

1

Phage Display Peptide Library Screening

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The Ph.D.-7 Phage Display Peptide Library kit was purchased from New England BioLabs, Inc. (Ipswich, MA, USA). Screening procedures were performed according to the manufacturer's protocol (version 1.0) with some modifications. Firstly, CHO and HO-8910 cells were digested with trypsin and the number of cells was adjusted to 1×107/ml. Subsequently, 100 µl CHO cells were transferred to an Eppendorf tube and 10 µl of the Ph.D.-7 Phage-Display Peptide Library was added, which initially contained 2×1011 plaque-forming units (pfu). The cells were incubated at 4°C for 2 h. A total of 200 µl organic solvent was added to the tube, which consisted of 180 µl dibutyl phthalate (DBP) and 20 µl cyclohexane (Beijing Yiqiangsheng Technology Co., Ltd., Beijing China). The tube was subsequently centrifuged at 10,000 × g for 10 min. Following centrifugation, the soluble fluid upper layer was pipetted into a fresh tube, which contained HO-8910 cells, and was incubated at 4°C for 3 h. The precipitate was transferred to a fresh tube, and 200 µl Luria-Bertani (LB) with E. coli ER2738 (mid-log phase) was added and incubated at 37°C for 30 min. Subsequently, phage was titrated and amplified, according to the manufacturer's instructions (New England BioLabs, Inc.; www.neb.com/protocols/2014/ 05/08/m13-titer-protocol). Finally, 5 rounds of in vitro reiterative biopanning were performed.
WANG L., HU Y., LI W., WANG F., LU X., HAN X., LV J, & CHEN J. (2016). Identification of a peptide specifically targeting ovarian cancer by the screening of a phage display peptide library. Oncology Letters, 11(6), 4022-4026.
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2

Phage Display Screening of Anti-ErbB2 Antibodies

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The PhD-7 phage display peptide library kit was purchased from New England BioLabs (Beverley, MA, USA). Biopanning of PhD-7 phage display peptide library with mouse anti-ErbB2 mAbs was performed according to the manufacturer's instructions.
Meng Y., Zheng L., Yang Y., Wang H., Dong J., Wang C., Zhang Y., Yu X., Wang L., Xia T., Zhang D., Guo Y, & Li B. (2016). A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors. Oncogenesis, 5(3), e211-.
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3

Isolation of CPMV Binding Peptides

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The Ph.D.-7 Phage Display Peptide Library Kit (New England Biolabs) with 109 unique sequences was used for isolation of CPMV binding peptides. 5 μg of CPMV was coated into each well of Nunc Maxisorp flat-bottom 96-well plates and incubated overnight at 4 °C. 109 pfu of the pre-blocked phage library (in 2% (w/v) bovine serum albumin; BSA) was added after the following blocking steps to remove BSA binders. The next day, CPMV was drained off and the well blocked with 2% (w/v) BSA for 1 h at room temperature (rt) with shaking at 800 rpm. The wells were washed thrice with Tris-buffered saline with 0.1% (v/v) Tween-20 (TBST) for 1 min per wash. The phage library was then added into the well and incubated for 30 min at rt with shaking at 800 rpm. The wells were washed thrice with TBST for 5 min per wash to remove unbound phages. The concentration of Tween-20 in TBST was increased from 0.1% to 0.3% and then 0.5% for each subsequent round to increase the selection stringency. Bound phages were eluted from CPMV by incubation with 0.2 M glycine–HCl (pH 2.2) for 18 min at rt and immediately neutralized by adding 1 M Tris–HCl buffer (pH 9.1). The eluted phages were subjected for amplification and titer according to the manufacturer’s protocol.
Chan S.K, & Steinmetz N.F. (2021). Isolation of Cowpea Mosaic Virus-Binding Peptides. Biomacromolecules, 22(8), 3613-3623.
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4

Phage Display Screening of FGFR2IIIc Binders

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The Ph.D. -7 phage display peptide library kit (New England BioLabs, Ipswich, MA, USA) was used for biopanning. Recombinant FGFR2IIIc extracellular domain protein (Sino Biological Inc., Beijing, China) was the screening target. All procedures were performed as previously described 76 . After three rounds, plaques containing the phage genome were sequenced to identify the phage peptide.
Zhao Y., Wang Q., Xie C., Cai Y., Chen X., Hou Y., He L., Li J., Yao M., Chen S., Wu W., Chen X, & Hong A. (2021). Peptide ligands targeting FGF receptors promote recovery from dorsal root crush injury via AKT/mTOR signaling. Theranostics, 11(20), 10125-10147.
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5

Biopanning for High-Affinity Protein Binders

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The recombinant cytosolic portion of DevS protein DevS201 (amino acids 377 to 578), was overexpressed and purified from E. coli BL21 harboring recombinant plasmid pSCS201 as described earlier [7 (link)]. The Ph.D.-7 Phage display peptide library kit (New England Biolabs Inc., Beverely, MA, USA) was screened by biopanning using the manufacturer’s instructions with a few modifications as outlined in Figure 1. Briefly, three rounds of panning were performed on a polystyrene 96-well micro titer plate wherein duplicate wells were coated with purified protein DevS201 -His6 (10 μg/well) and the phage library (2 × 1011 phages) was incubated with the immobilized protein to allow binding of the phage particles. Decreasing the time of incubation with DevS201 protein and increasing the percentage of Tween-20 in the washing buffer in each successive round of panning achieved the stringent selection of high affinity binder phages. Three rounds of panning were performed in all and each time the phages in the glycine elution (0.2 M glycine, pH 2.2) were amplified and used as an input for the next round of panning. The titer of phages in the various elutions was determined according to the manufacturer’s instructions and the input phage number was maintained during each round of panning.
Kaur K., Taneja N.K., Dhingra S, & Tyagi J.S. (2014). DevR (DosR) mimetic peptides impair transcriptional regulation and survival of Mycobacterium tuberculosis under hypoxia by inhibiting the autokinase activity of DevS sensor kinase. BMC Microbiology, 14, 195.
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6

Phage Display Peptide Library Screening

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15 μg of MCA 100 μg/ml in 0.1 M NaHCO3 pH 8,6 were incubated in 96-well plates (Costar EIA/RIA High binding) at 4 °C o/n. Wells were blocked with 150 μl of 5 mg/ml BSA in 0.1 M NaHCO3 pH 8,6 for 1 h at 4 °C, washed six times with TBST (50 mM Tris-HCl pH 7,5, 150 mM NaCl, 0,1% Tween 20) and incubated with 2 × 1011 phages from Ph.D.- 7 Phage Display Peptide Library Kit (New England Biolabs, Ipswich, MA, USA) in 100 μl of TBST for 1 h at room temperature. Unbound phages were removed by 10 washing steps in TBST and bound phages were recovered by incubation with 200 μl of exponentially growing (OD600=0,5) E. coli (ER2738) for 5 min at room temperature. Tittering and amplification were performed as recommended.
Peña M.S., Cabral G.C., Fotoran W.L., Perez K.R, & Stolf B.S. (2017). Metacaspase-binding peptide inhibits heat shock-induced death in Leishmania (L.) amazonensis. Cell Death & Disease, 8(3), e2645-.
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7

Phage Display Selection of HORMA Peptides

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For phage display, we used a commercial assay kit (Ph.D.-7 Phage Display Peptide Library Kit, New England Biolabs) and followed the recommended protocol for “solution phase panning with affinity bead capture” with the following modifications. We used His6-tagged Ec HORMA, Pa HORMA2, and Pa HORMA3 as bait proteins. For affinity purification, we diluted 2 μL of bait protein at 0.2 mM and 10 μL phage library into 200 μL volume in TBST (50mM Tris pH7.5, 150mM NaCl, plus 0.1% Tween (first round) or 0.5% Tween (subsequent rounds)). After a 20 minute incubation at room temperature, 50 μL of magnetic Ni-NTA beads (Qiagen; pre-blocked with blocking buffer (0.1M NaHCO3, pH 8.6, 5mg/mL BSA, 0.02% NaN3) and washed 3x with TBST) were added, and the mixture was incubated a further 15 minutes at room temperature. Beads were washed 10x with wash buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, plus 0.1% Tween (first round) or 0.5% Tween (subsequent rounds)). For elution, 1 mL elution buffer (0.2M Glycine-HCl, pH 2.2, 1mg/mL BSA) was added, incubated for 10 minutes at room temperature, then neutralized the eluate by adding 150 μL Tris-HCl pH 9.1. Eluted phage were titered and amplified according to standard protocols, and the selection was repeated, Following four rounds of selection, phages were isolated and the variable peptide region sequenced for at least 20 individual clones.
Ye Q., Lau R.K., Mathews I.T., Birkholz E.A., Watrous J.D., Azimi C.S., Pogliano J., Jain M, & Corbett K.D. (2020). HORMA domain proteins and a Trip13-like ATPase regulate bacterial cGAS-like enzymes to mediate bacteriophage immunity. Molecular cell, 77(4), 709-722.e7.
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8

Phage Display for Glioblastoma Stem Cell Targeting

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In vitro and in vivo phage display biopanning was performed using a Ph.D.-7 Phage Display Peptide Library Kit (New England BioLabs, Ipswich, MA). The first step of the in vitro biopanning was a negative selection of the library against extracellular matrix and DGCs to remove non-GSC binding peptides. The peptide library was then screened against GSCs for four rounds of biopanning. Phage clones were then isolated through bacterial infection and peptide sequences were isolated by sequencing. In vivo biopanning was performed by intravenously injecting the phage library in NSG mice bearing intracranial GBM. Following circulation of the peptide library for 24 hours, tumors were harvested, tumor cells were lysed, then bound phage peptides were isolated, purified, and transduced into E. coli for phage clone isolation and sequenced. The in vivo phage display performed against subcutaneous GBM was performed as previously described (22 (link)).
Potez M., Snedal S., She C., Kim J., Thorner K., Tran T.H., Ramello M.C., Abate-Daga D, & Liu J.K. (2023). Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells. Frontiers in Oncology, 13, 1124272.
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9

Phage Display Peptide Library Kit

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The PhD-7 Phage Display Peptide Library Kit (New England BioLabs, USA) was used according to methodology previously described by Wu et al. (2006) (link). The technology consists of a randomized linear 7-mer peptides fused to a minor coat protein (pIII) of M13 phage.
Buzatti A., Fernandez A.D., Arenal A., Pereira E., Monteiro A.L.G, & Molento M.B. (2018). Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library. Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria, 27(2).
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