Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance.
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.
Yano
Kazuyoshi
K
School of Bionics, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan.
Yoshino
Tetsuya
T
Shionoya
Makoto
M
Sawata
Shinya Y
SY
Ikebukuro
Kazunori
K
Karube
Isao
I
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D019151:Peptide Library
D019151:Peptide Library; D000469:Alkaline Phosphatase