BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3409

Peptides:

Sequences

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)

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 12900423

Target Name:

Chymase (EC:3.4.21.39)

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

X9 T7 phage display library

Affinity Measurement Method:

Affinity Measurement Description:

Brief Description:

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.

BiopanningDataSet Comments:

Curator:

Bifang He

Curation Date:

2019-05-17

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