Objective: Recently, PHAGE display libraries have received enormous attention for identification and isolation of pharmaceutical molecules with diagnostic and therapeutic properties. PEPTIDE libraries are known as one of the most important and widely used types of PHAGE display libraries. In the current study, we aimed to screen the Ph.D. TM-7 PHAGE display PEPTIDE LIBRARY through biopanning for the identification of human colon adenocarcinoma-binding PEPTIDE ligands.Methods: Three rounds of biopanning were performed on SW480 as the target cell and fibroblast (HF-SF-PI3), AGS, KYSE-30 and Huh-7 as control cells. The displayed PEPTIDE-encoding regions in the genome of SW480-binding PHAGEs obtained from the final round of panning were amplified by plaque-PCR and subsequently sequenced. Bioinformatic tools were used to determine the sequence of target cell-binding PEPTIDEs and further characterization of these PEPTIDEs.Results: Biopanning of the PHAGE LIBRARY led to the enrichment of several PEPTIDEs among which the PEPTIDE with sequence “HAMRAQP” was the most dominant. Bioinformatic analysis of the isolated PEPTIDEs indicated that they are not target unrelated PEPTIDEs (TUP).Conclusion: The PEPTIDEs, in particular those with the highest frequency, due to having the capability of specific binding to SW480 cells represent the potential for use in targeting of therapeutic genes and drugs to colon cancer cells.

PHAGE display PEPTIDE libraries (PDPLs) harbor a large abundance of PEPTIDEs displayed on the surface of filamentous PHAGEs, representing the potential to be used for a wide variety of applications. Over the recent years, PDPLs have been used for identifying targeting PEPTIDEs to a variety of targets. Screening of PDPLs on human cells has proven to be a highly-valued venue for isolating PEPTIDEs that recognize target cells with high affinity and specificity. Prostate cancer is one of the most common causes of cancer-associated death in men. Studies have indicated that targeting PEPTIDEs to prostate cancer cells represent potential to be used as valuable diagnostic and therapeutic tools. In the current study, we aim to isolate PEPTIDEs targeting to PC3 cells (human prostate cancer cells). A heptaPEPTIDE PDPL was used through biopanning to isolate PEPTIDEs binding specifically to PC3 cells. The panning was performed on PC3 and control cells including 5637, Huh-7, SW480, AGS and human normal cells. Polyclonal-PHAGE ELISA was exploited to evaluate the process of enrichment during biopanning. Subsequently, PHAGE clones were randomly picked out from titer plates, amplified by using plaque-PCR and their genomic DNA was sequenced. Bioinformatic analysis was conducted for further characterization of isolated PEPTIDEs. Several rounds of panning resulted in the enrichment of PEPTIDEs. The PEPTIDE identified through biopanning can be considered as a potential specific binder to PC3 cells. Further analysis of this PEPTIDE is required to show its capacity for targeted delivery of various gene and drug delivery vehicles into prostate cancer cells.

Objective: Prostate cancer is the second cause of cancer-associated death in men. In recent years, targeted therapy for cancer has attracted the attention of researchers.Targeted therapy leads to a decrease in drug adverse effects. Studies indicate that targeting PEPTIDEs for cancer cells represent valuable tools for diagnostics and therapeutics. Recently, PHAGE display PEPTIDE libraries have been used to identify target PEPTIDEs to a variety of cancer cells. In the current study, we aim to isolate PEPTIDEs that target PC3 cells (human prostate adenocarcinoma cells).Methods: Four rounds of subtractive panning on control cells that included 5637 (bladder), Huh-7 (liver), SW480 (colon), AGS (stomach) and human fibroblast normal in addition to four rounds of positive panning on PC3 (target cell) were performed.Polyclonal PHAGE ELISA was used to evaluate the process of enrichment during biopanning. Subsequently, PHAGE clones were randomly selected from titer plates, amplified by plaque-PCR, and their genomic DNA was sequenced. We conducted bioinformatic analysis for further characterization of the isolated PEPTIDEs.Results: Several rounds of panning resulted in the enrichment of a number of PEPTIDEs.The results of polyclonal PHAGE ELISA indicated that the biopanning process was successful. In silico analysis showed the presence of several consensus amino acid motifs in the PEPTIDEs.Conclusion: The PEPTIDEs identified through biopanning can be considered as potential specific binders to PC3 cells. PEPTIDEs with specificity binding to target cells can be used for targeted gene and drug delivery to malignant tumor cells. Further analyses of these PEPTIDEs are required to show their capacity for targeted delivery of various genes and drugs into prostate cancer cells.

The commercial availability of random PEPTIDE libraries displayed on the M 13 PHAGE increased their use for studies on epitope identification, enzyme inhibitors and receptor ligands. We planed two experiments for selection of PEPTIDEs. First, with sheep antibodies, the positive selector was IgG, prepared on Protein G columns from a pool of 11 sheep protected with a vaccine prepared from larvae of Lucilia cuprina, against blowfly strike and the negative selectors were the IgG from the same sheep before vaccination and IgG from vaccinated sheep which were not protected. Four rounds of positive and negative selection were done respectively. In the second experiment, human IgG again prepared on new Protein G columns. Four rounds of positive selection with either IgG from MS or schizophrenia patients were done. They were alternated with two negative selections of schizophrenia IgG on the MS associated PEPTIDEs and vice versa and two negative selections on each with the IgG from people who had died from non-nervous system diseases as control. After the fourth negative selection of each experiment, the PHAGE were amplified and random clones were picked for nucleotide sequencing. A total of 44 PEPTIDEs were sorted with the PILEUP program. From those, 15 PEPTIDEs had a large number with either 4 or 5 methionines.

The commercial availability of random PEPTIDE libraries displayed on the M13 PHAGE is increasing their use for studies on epitope identification, enzyme inhibitors, receptor ligands, etc. In this study two experiments where planned for selection of PEPTIDEs. First with sheep antibodies, the positive selector was IgG, prepared on Protein G column from a pool of 11 sheeps immunized, with a vaccine prepared from larvae of Lucilia cuprina, against blowfly strike. The negative selector was IgG from the same sheeps before vaccination and IgG from vaccinated non-immunized sheeps. Four rounds of positive and negative selections with IgG from sheep were done respectively. In the second experiment using human IgG prepared on a fresh Protein G column, again four rounds of positive selection with either IgG from MS and schizophrenia patients were performed. This assay was alternated with two negative selections of schizophrenia IgG on MS-associated PEPTIDEs and vice versa and two negative selections on each with a control IgG. After the fourth negative selection in each experiment, the PHAGE was amplified and random clones were picked for nucleotide sequencing. A total of 44 PEPTIDEs were sorted with the PILEUP program and there was a large number of 12-mer PEPTIDEs containing either 4 or 5 methionine residues