20.109(S11):TR Yellow Research Proposal

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(Selection of peptide binding sequences to human alpha thrombin using mRNA display evolution)
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===Background===
===Background===
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By harnessing the power of natural selection to modify a protein’s properties through directed evolution, a library of mutant proteins must first be created. If this process were performed in vivo, the size of the library would have to be limited to between 10^4-10^9 members, and if it were performed in vitro, the size of the library could be expanded to over 10^12 members. The protocol described in this proposal, called “mRNA display,” uses mRNA-displayed proteins, which are proteins that have the strand of mRNA that codes for the protein covalently attached. This creates a link between genotype (the mRNA strand) and phenotype (the attached protein), and therefore enables direct amplification of the desired sequence. The linkage enables several rounds of amplification and selection in libraries larger than 10^12 in one experiment.  
+
By harnessing the power of natural selection to modify a protein’s properties through directed evolution, a library of mutant proteins must first be created. If this process were performed in vivo, the size of the library would have to be limited to between 10^4-10^9 members, and if it were performed in vitro, the size of the library could be expanded to over 10^12 members. The protocol described in this proposal, called “mRNA display,” uses mRNA-displayed proteins, which are proteins that have the strand of mRNA that codes for the protein covalently attached. This creates a link between genotype (the mRNA strand) and phenotype (the attached protein), and therefore enables direct amplification of the desired sequence. The linkage enables several rounds of amplification and selection in libraries larger than 10^12 in one experiment. (Seelig-see references)
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mRNA display has been used to isolate small, functional peptides that bind to and inhibit human alpha thrombin. The sequences of these peptides were analyzed and compared, and out of the 45 sequences that bound to thrombin, more 60% of the sequences contained the conserved four amino acid sequence motif, DPGR. From this, it was concluded that this amino acid sequence plays a crucial role in the binding of the peptide sequence to alpha thrombin.  
+
mRNA display has been used to isolate small, functional peptides that bind to and inhibit human alpha thrombin. The sequences of these peptides were analyzed and compared, and out of the 45 sequences that bound to thrombin, more 60% of the sequences contained the conserved four amino acid sequence motif, DPGR. From this, it was concluded that this amino acid sequence plays a crucial role in the binding of the peptide sequence to alpha thrombin. (Raffler et al- see references)
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Revision as of 12:25, 26 April 2011

Contents

Selection of peptide binding sequences to human alpha thrombin using mRNA display evolution

Stephanie Fung, Shireen Rudina

Statement of Purpose

Using directed evolution, we want to find peptides that bind and hopefully inhibit human alpha thrombin. Previous research has shown the GGF motif to exist in all sequences that were found to bind to thrombin, so we want to generate a library based on this motif, and then use mRNA display evolution to find the best possible binder through multiple iterations of library generation and selection. Finding new binders and inhibitors of alpha thrombin can serve as candidates for new anti-thrombotic therapeutics.


Background

By harnessing the power of natural selection to modify a protein’s properties through directed evolution, a library of mutant proteins must first be created. If this process were performed in vivo, the size of the library would have to be limited to between 10^4-10^9 members, and if it were performed in vitro, the size of the library could be expanded to over 10^12 members. The protocol described in this proposal, called “mRNA display,” uses mRNA-displayed proteins, which are proteins that have the strand of mRNA that codes for the protein covalently attached. This creates a link between genotype (the mRNA strand) and phenotype (the attached protein), and therefore enables direct amplification of the desired sequence. The linkage enables several rounds of amplification and selection in libraries larger than 10^12 in one experiment. (Seelig-see references)

mRNA display has been used to isolate small, functional peptides that bind to and inhibit human alpha thrombin. The sequences of these peptides were analyzed and compared, and out of the 45 sequences that bound to thrombin, more 60% of the sequences contained the conserved four amino acid sequence motif, DPGR. From this, it was concluded that this amino acid sequence plays a crucial role in the binding of the peptide sequence to alpha thrombin. (Raffler et al- see references)


Protocol

  1. Generate a library based around the DPGR motif
  2. mRNA display evolution to select for binders
  3. Identify motif
  4. Generate new library based around new motif
  5. Iterate.
  6. Test for thrombin activity. Assay?

Resources

mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries

A Novel Class of Small Functional Peptides that Bind and Inhibit Human α-Thrombin Isolated by mRNA Display

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