Author : Sheila J. Sang
Publisher :
ISBN 13 :
Total Pages : 132 pages
Book Rating : 4.:/5 (896 download)
Book Synopsis The Use of Phage Display to Identify Specific Peptide Ligands by : Sheila J. Sang
Download or read book The Use of Phage Display to Identify Specific Peptide Ligands written by Sheila J. Sang and published by . This book was released on 2014 with total page 132 pages. Available in PDF, EPUB and Kindle. Book excerpt: Phage display allows the expression of peptides on filamentous phage when the peptide of interest is fused to the gene for a virus coat protein. The goal of this project was to use phage display to produce peptide molecules that bind specifically to human serum albumin. An M13 library displaying random linear heptapeptides linked to coat protein pIII (Ph.D.-7[trademark], New England BioLabs) was amplified by infecting E. coli host strain ER2738 to produce phage library stocks Amp LA (2.1x1012 pfu/ml) and Amp LS (1.0x109 pfu/ml). The amplified phage were titered on LB plates containing X-gal, IPTG, and tetracycline to ensure expression of the F pilus. Three rounds of biopanning on HSA-coated polyvinyl chloride plates and amplification of the virus was performed (Amp P3A and Amp P3S). Individual plaques were picked and amplified to produce clones (HSA1- HSA8). We developed a phage concentration ELISA to test for the quantity of phage needed to detect binding in an ELISA using peroxidase-conjugated antibodies against M13. Phage were diluted in sodium carbonate to bind the protein to the plate, followed by block and anti-M13-PO. It was found that a concentration greater than 106 pfu/ml was needed to give a positive M13 ELISA. We then tested the conditions needed to produce an ELISA that measures phage binding to a specific ligand (M13 ELISA). Three different plates were tested. Although MaxiSorp® plates are thought to give a good signal and more consistent data, we found that specific binding of Amp LA was best when using polyvinyl chloride plates. The influence of blocking buffer (ovalbumin and casein) was also tested. Casein block and sample buffer gave a good signal in the presence of HSA with the least non-specific binding. These studies demonstrate that phage display is an effective method for producing HSA-binding peptides.