Objective To use phage display technique to screen for small polypeptides that specifically bind to MDA-MB-468 cells.Methods A random heptapeptide phage display library was used for in vitro screening against target M...Objective To use phage display technique to screen for small polypeptides that specifically bind to MDA-MB-468 cells.Methods A random heptapeptide phage display library was used for in vitro screening against target MDA-MB-468 cells.SC1180 cells were used for subtractive selection.High-affinity phage DNA was extracted,and peptides were sequenced.Results(1)The original library capacity of the polypeptide library was 2×10^13 pfu/mL,and phage titer was determined over 4 rounds.The average library capacity was 1.8×10^13 pfu/mL.(2)Subtractive screening showed that the phage library volume of each round was 1.8×10^12 pfu/mL,and that there was an enrichment effect in each subsequent round.Screening was stopped after the fourth round.(3)PCR results showed that the size of 39 products(78.0%)and 11 products(22%),were 300 bp and 258 bp,respectively.Thirty positive phages were selected for DNA extraction and sequencing,and the corresponding amino acid sequence was LMTRXSK.The sequence had no homology with known genes or proteins.Conclusion Using the phage display technique,we identified that the short polypeptide,LMTRXSK,specifically binds MDA-MB-468 human breast cancer cells.展开更多
With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measu...With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.展开更多
基金Supported by a grant from the Jiangsu Provincial Health and Family Planning Commission(No.H201640)
文摘Objective To use phage display technique to screen for small polypeptides that specifically bind to MDA-MB-468 cells.Methods A random heptapeptide phage display library was used for in vitro screening against target MDA-MB-468 cells.SC1180 cells were used for subtractive selection.High-affinity phage DNA was extracted,and peptides were sequenced.Results(1)The original library capacity of the polypeptide library was 2×10^13 pfu/mL,and phage titer was determined over 4 rounds.The average library capacity was 1.8×10^13 pfu/mL.(2)Subtractive screening showed that the phage library volume of each round was 1.8×10^12 pfu/mL,and that there was an enrichment effect in each subsequent round.Screening was stopped after the fourth round.(3)PCR results showed that the size of 39 products(78.0%)and 11 products(22%),were 300 bp and 258 bp,respectively.Thirty positive phages were selected for DNA extraction and sequencing,and the corresponding amino acid sequence was LMTRXSK.The sequence had no homology with known genes or proteins.Conclusion Using the phage display technique,we identified that the short polypeptide,LMTRXSK,specifically binds MDA-MB-468 human breast cancer cells.
文摘With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.
