Development of peptide-like library

Peptide libraries are important in peptide based drug discovery and investigations of biological targets. Different chemical and biological methods have been developed to screen and decode peptide libraries. mRNA display technology is a biological method that utilizes in vitro translation systems to...

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Main Author: Zhou, Miao
Format: Others
Language:English
Published: University of British Columbia 2009
Online Access:http://hdl.handle.net/2429/7232
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spelling ndltd-LACETR-oai-collectionscanada.gc.ca-BVAU.-72322013-06-05T04:17:19ZDevelopment of peptide-like libraryZhou, MiaoPeptide libraries are important in peptide based drug discovery and investigations of biological targets. Different chemical and biological methods have been developed to screen and decode peptide libraries. mRNA display technology is a biological method that utilizes in vitro translation systems to build peptide libraries, and the screened peptides are decoded by their covalently attached oligonucleotide templates. Like other biological methods, mRNA display technology can generate peptide libraries with high fidelity and speed but the chemical repertoire is limited mostly to natural amino acids. Our research goal is to expand the chemical repertoire of mRNA display technology by using it in combination with an in vitro reconstituted translation system, and build peptides containing stabilizing chemical features such as N-methyl amino acids that would make peptides more proteolytic resistant and drug-like. Using a streptavidin-dependent gel shift assay, the unnatural amino acids, N-methyl-L-alanine, N-methyl-L-glutamate, N-methyl-L-ornithine, L-abrine, t-butyl-L-glycine, biocytin, and sarcosine were shown to be able to incorporate successively into hexa-peptides in the mRNA display format. The peptides containing unnatural amino acids were shown to be more resistant to proteinase K proteolysis than peptides containing only the natural amino acid L-alanine. Several mRNA libraries were made in accordance with the mRNA display format. Repeated E. coli in vitro reconstituted translations of these mRNA libraries with varying translation conditions fail to yield peptides of significant lengths. However, a library of tetra-peptides was synthesized in the absence of mRNA display format. Thus, the puromycin linker required by mRNA display technology seemed to interfere with proper translations in the E. coli in vitro reconstituted translation system. Experiments showed that E. coli in vitro reconstituted translation system is slow at making full-length peptides in the context of mRNA display technology and a majority of products are prematurely terminated translation products, which could be due to slow peptidyl formation in the E. coli in vitro reconstituted translation system, or the result of undesirable interaction between the E. coli ribosome and puromycin linker that interfere with normal translation. This slow translation kinetic issue needs to be addressed with further experiments in order to successfully pursue the development of this technology.University of British Columbia2009-04-16T18:40:55Z2009-04-16T18:40:55Z20092009-04-16T18:40:55Z2009-05Electronic Thesis or Dissertation2872595 bytesapplication/pdfhttp://hdl.handle.net/2429/7232eng
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language English
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description Peptide libraries are important in peptide based drug discovery and investigations of biological targets. Different chemical and biological methods have been developed to screen and decode peptide libraries. mRNA display technology is a biological method that utilizes in vitro translation systems to build peptide libraries, and the screened peptides are decoded by their covalently attached oligonucleotide templates. Like other biological methods, mRNA display technology can generate peptide libraries with high fidelity and speed but the chemical repertoire is limited mostly to natural amino acids. Our research goal is to expand the chemical repertoire of mRNA display technology by using it in combination with an in vitro reconstituted translation system, and build peptides containing stabilizing chemical features such as N-methyl amino acids that would make peptides more proteolytic resistant and drug-like. Using a streptavidin-dependent gel shift assay, the unnatural amino acids, N-methyl-L-alanine, N-methyl-L-glutamate, N-methyl-L-ornithine, L-abrine, t-butyl-L-glycine, biocytin, and sarcosine were shown to be able to incorporate successively into hexa-peptides in the mRNA display format. The peptides containing unnatural amino acids were shown to be more resistant to proteinase K proteolysis than peptides containing only the natural amino acid L-alanine. Several mRNA libraries were made in accordance with the mRNA display format. Repeated E. coli in vitro reconstituted translations of these mRNA libraries with varying translation conditions fail to yield peptides of significant lengths. However, a library of tetra-peptides was synthesized in the absence of mRNA display format. Thus, the puromycin linker required by mRNA display technology seemed to interfere with proper translations in the E. coli in vitro reconstituted translation system. Experiments showed that E. coli in vitro reconstituted translation system is slow at making full-length peptides in the context of mRNA display technology and a majority of products are prematurely terminated translation products, which could be due to slow peptidyl formation in the E. coli in vitro reconstituted translation system, or the result of undesirable interaction between the E. coli ribosome and puromycin linker that interfere with normal translation. This slow translation kinetic issue needs to be addressed with further experiments in order to successfully pursue the development of this technology.
author Zhou, Miao
spellingShingle Zhou, Miao
Development of peptide-like library
author_facet Zhou, Miao
author_sort Zhou, Miao
title Development of peptide-like library
title_short Development of peptide-like library
title_full Development of peptide-like library
title_fullStr Development of peptide-like library
title_full_unstemmed Development of peptide-like library
title_sort development of peptide-like library
publisher University of British Columbia
publishDate 2009
url http://hdl.handle.net/2429/7232
work_keys_str_mv AT zhoumiao developmentofpeptidelikelibrary
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