BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3560

Peptides:

Sequences

Affinity Values

CQGPTFKCDAIWREC(20)
CVPQPDTCAKIDPRC(8)
CLLQQPVCGEWNPKC(7)
CYAAQGGCLRDWTRC(4)
CYAAQGGCLRDWTRC(3)
CYAINQRCLQEWSRC(3)
CFADGSNCVVEWSEC(3)
CIVQQGLCHEWNPRC(3)
CVWLQATCTRSWSGC(3)
CRPQPDTCVSLSGEC(2)
CVPQPDTCANMDPEC(1)
CKPQPDTCLHTTGKC(1)
CYAAKGGCQPNWTQC(1)
CYQSWPVCQAWNPRC(1)
CQHAGAVCHWWNPRC(1)
CNQDLMVCRFWNSRC(1)
CRKMAEDCGWGVLVC(1)
CKKVSMSCGWGEAVC(1)
CRERMAVCGWGVQVC(1)
CNWQTQVCVRDWLGC(1)
CSWQQGECTRTWGGC(1)

[3.4±0.2]
[>300]
[>300]
[~100]
[>300]
[~250]
[N.D.]
[>300]
[~300]
[>300]
[>300]
[N.D.]
[~100]
[>300]
[>300]
[>300]
[>300]
[>300]
[>300]
[~300]
[>300]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 31251434

Target Name:

72 kDa type IV collagenase

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

CX6CX6C phage display library

Affinity Measurement Method:

Fluorescence

Affinity Measurement Description:

The inhibitory activity of bicyclic peptides was determined by measuring sidual protease activity with a fluorogenic substrate. For the FS-6 substrate, excitation was measured at 325 nm and emission at 400 nm. For the OMNIMMP® RED substrate, excitation was measured at 545 nm and emission at 576 nm. The inhibition constants (Ki) were calculated according to the equation of Cheng and Prusoff Ki = IC50/(1+([S]0/Km) wherein IC50 is the peptide concentration at which 50% of the enzymaticactivity is inhibited, [S]0 is the total substrate concentration, and KM is the Michaelis-Menten constant. Ki (μM) values were shown.

Brief Description:

Bacterial cells of a phage library glycerol stock were added to a 2 L falcon flask containing 500 ml of 2YT/chloramphenicol (30 μg/ml) medium to obtain an OD600 of 0.1. The culture was shaken (200 rpm) for 16 hr at 30°C. Bacterial cells were pelleted by centrifugation at 16,000 g and 4°C for 30 min, and the phage in the supernatant were purified by PEG-precipitation as follows. Briefly, 0.2 volumes of PEG solution (20% (w/v) polyethylene glycol 6000, 2.5 M NaCl) was added to the supernatant, mixed, incubated on ice for 30 min, and centrifuged at 2,700 g and 4°C for 30 min. PEG-purified phage, typically 1011–1012 t.u. (transducing units) were resuspended in 20 ml of 20 mM NH4HCO3, 5 mM EDTA, pH 8.0. Disulfide bridges were reduced by addition of 1 mM of TCEP and incubation at 42°C for 1 hr. The concentration of TCEP was subsequently reduced by repetitive concentration and dilution steps with reaction buffer (20 mM NH4HCO3, 5 mM EDTA, pH 8.0, degassed) in a Vivaspin-20 filter (MWCO of 10,000, Sartorius-Stedim Biotech GmbH). The volume of the phage solution was adjusted to 32 ml with reaction buffer and 8 ml of 50 μM tris-(bromomethyl)benzene (TBMB) in acetonitrile (MeCN) were added to obtain a final TBMB concentration of 10 μM. The reaction was incubated at 30°C for 1 hr before non-reacted TBMB was removed by precipitation of the phage with 0.2 volumes of 20% (w/v) polyethylene glycol 6000, 2.5 M NaCl on ice and centrifugation at 2,700 g and 4°C for 30 minutes. The phage pellet was resuspended in 3 ml of washing buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2). Biotinylated active MMP-2 (180 μg) was added to 50 μl magnetic streptavidin beads (Dynabeads M-280 from Invitrogen Dynal Biotech AS) in washing buffer and incubated on a rotating wheel for 10 min at room temperature (RT). The magnetic beads were then washed with 0.5 ml washing buffer and incubated for 30 min at RT with 0.5 ml washing buffer containing 1% (w/v) BSA and 0.1% (v/v) Tween 20. At the same time the chemically modified phage (typically e10–e11 t.u. dissolved in 3 ml of washing buffer) were blocked by addition of 1.5 ml of washing buffer containing 3% (w/v) BSA and 0.3% (v/v) Tween 20 for 30 minutes. The blocked beads/target protein mixture (0.5 ml) and phage (4.5 ml) were mixed together and incubated for 30 minutes on a rotating wheel at room temperature. The beads were washed eight times with washing buffer containing 0.1% (v/v) Tween 20 and twice with washing buffer. The phage were eluted by incubation with 100 μl of 50 mM glycine, pH 2.2 for 5 minutes (pH-dependent elution), and then transferred to 50 μl of 1 M Tris-Cl, pH 8.0 for neutralization. The eluted phage were incubated with 30 ml of TG1 cells at OD600 of 0.4 for 90 minutes at 37°C, and the cells were plated on large 2YT/chloramphenicol (30 μg/ml chloramphenicol) plates. The second and third round of panning were performed following the same procedure but using in the second round neutravidin-coated magnetic beads instead of streptavidin to prevent the enrichment of streptavidin-specific peptide binders. Neutravidin beads were prepared by reacting 0.8 mg neutravidin (Pierce) with 0.5 ml of tosyl-activated magnetic beads (2e9 beads/ml; Dynabeads M-280, Invitrogen Dynal Biotech AS) according to the supplier's instructions.

BiopanningDataSet Comments:

The abundant peptide M21 (ACQGPTFKCDAIWRECG) inhibited MMP-2 with a Ki of 3.4±0.2 μM. An alanine scan of M21 revealed that the first four amino acids of the C-terminal ring, Asp-Ala-Ile-Trp, were most important for the binding.

Curator:

Chunchao Pu

Curation Date:

2020-12-29

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