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Searching Protease Inhibitors by a Phage Display Kunitz‐Type Library.
Author(s) -
Silva Gustavo Felippe,
Lourdes Borba Magalhães Maria,
Moraes Julio Cesar,
Echeverri Lina Maria Salazar
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.530.31
Subject(s) - phage display , phagemid , protein engineering , peptide library , protease , mutagenesis , microbiology and biotechnology , scaffold protein , biology , chemistry , computational biology , biochemistry , gene , peptide sequence , bacteriophage , enzyme , mutation , escherichia coli , peptide , signal transduction
Phage Display is a powerful tool for identification of protein ligands against a myriad of targets. In our study, we selected a small protein member from the Kunitz family to serve as a scaffold in the MIII 13 phage coat protein. The protein, originally a scorpion toxin that inhibits a potassium‐dependent voltage channel, has 33 residues composed of two beta sheets and an alpha helix stabilized by disulfide bonds. After cloning the toxin gene into an M13 phagemid, we used Kunkel's mutagenesis, an additional uracil‐DNA glycosylase treatment, and rolling circle amplification (RCA) procedures to produce a synthetic Kunitz‐type phage display library. We drove the mutations in specific residues within two specific loops as well as within the alpha helix of the scaffold protein, keeping the cysteines intact. We were able to produce a library of over a million different proteins. To validate the ability of this library to bind specific targets, trypsin was used as target. Since other studies have proved Kunitz's natural proteins as protease inhibitors, we expect our displayed Phage protein library to generate new potent and specific protease inhibitors for medical as well as biotechnological purposes. Support or Funding Information This work was supported by “State University of Santa Catarina” (UDESC) and Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .