Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance

• Published in: Biosensors & bioelectronics Vol. 18; no. 10; pp. 1201 - 1207
• Main Authors: Yano, Kazuyoshi, Yoshino, Tetsuya, Shionoya, Makoto, Sawata, Shinya Y., Ikebukuro, Kazunori, Karube, Isao
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01-09-2003; Elsevier Science
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - metabolism; Amino Acid Sequence; Bacteriophage M13 - genetics; Bacteriophage M13 - metabolism; Base Sequence; Biological and medical sciences; Biosensors; Biotechnology; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli - genetics; Escherichia coli genome; Fundamental and applied biological sciences. Psychology; Genomic Library; M13; Methods. Procedures. Technologies; Molecular Sequence Data; Peptide Library; Phage display; Surface plasmon resonance; Various methods and equipments; Alkaline phosphatase; Phage display; Surface plasmon resonance; Escherichia coli genome; M13; Enzyme; Escherichia coli; Phosphoric monoester hydrolases; Esterases; Surface plasmon; Phage M13; Protein; Virus; Binding capacity; Biosensor; Bacteria; Hydrolases; Resonance; Genome; Phage; Enterobacteriaceae
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/S0956-5663(03)00082-4

A novel phage library has been prepared using the
Escherichia coli genome digested with three restriction enzymes. The resulting DNA fragments were ligated to the expression vector pCANTAB5 to obtain the library of recombinant M13 phages displaying relatively long exogenous peptides. The library was screened to isolate recombinant phages with high affinity to alkaline phosphatase (AP) from calf intestine. After four rounds of panning three phages (AP1, AP2 and AP3) were shown to have specific binding properties toward AP by enzyme-linked immunosorbent assay. The phages were further characterized by surface plasmon resonance (SPR). Among the three phages AP3 bound the AP-immobilized sensor chip most and caused the highest resonant angle shift. The sensor response decreased with the decrease of the concentration of AP3 added. Furthermore, displacement of AP3 from the AP-immobilized sensor chip was observed upon injection of AP solution to the SPR system, whereas injection of bovine serum albumin solution led to the great increase of the sensor response. This result indicates the specific binding of AP3 to AP.