Details

Autor(en) / Beteiligte

• Yun, Soi
• Lee, Sangah
• Park, Jong Pil
• Choo, Jaebum
• Lee, E.K.

Titel

Modification of phage display technique for improved screening of high-affinity binding peptides

Ist Teil von

• Journal of biotechnology, 2019-01, Vol.289, p.88-92

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2019

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• •Biopanning performance can be improved by contacting a target with phage library before its immobilization to a solid matrix.•Peptide’s binding affinity can be dramatically enhanced by dimerization linked with a beta-hairpin-structured peptide.•A modified phage screening combined with affinity maturation can be applied to prepare antibody-mimicking peptides. The phage display technique is a combinatorial technology in which random peptides are displayed on the surface of the phage; it is widely used to identify high-affinity peptides that bind to a target protein. However, this technique presents several problems due to non-specific binding of the phages and steric hindrance caused by blocking agents. To overcome these problems, we tested two modified methods and compared their screening performance with that of the conventional method. We used poly-His-tagged human epidermal growth factor receptor 2 (HER2) as a target protein and silica-coated magnetic particles (MPs) with an immobilized nickel-nitrilotriacetic acid ligand as a solid matrix. Modified method #1 (#M1) included two negative selection steps against a blocking agent (bovine serum albumin) and nude Ni-NTA MPs, after the positive selection step using immobilized HER2 on MPs in the absence of BSA. Modified method #2 (#M2) allowed the binding of phages and HER2 in solution prior to immobilizing HER2 on the MP surface. The negative selection procedure was the same between them. The binding affinity of the phages screened by #M1 and #M2 was evaluated by phage ELISA. Two phages from #M2 (#M2-4 and 5) showed the highest binding, and between them #M2-5 was selected for affinity maturation by inserting a hairpin-structured peptide between the two #M2-5 peptides. SPR study showed the HER2-binding affinity was indeed improved by ca. 2000-fold to become comparable with that of the intact antibody (trastuzumab). The modified biopanning methods combined with the dimerization maturation can be an effective way to develop antibody-mimicking peptides for affinity binding.