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Title: Method of generating ploynucleotides encoding enhanced folding variants

Abstract

The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

Inventors:
; ;
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1354812
Patent Number(s):
9,637,528
Application Number:
12/286,967
Assignee:
Los Alamos National Security, LLC LANL
DOE Contract Number:  
AC52-06NA25396
Resource Type:
Patent
Resource Relation:
Patent File Date: 2008 Oct 02
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Bradbury, Andrew M., Kiss, Csaba, and Waldo, Geoffrey S. Method of generating ploynucleotides encoding enhanced folding variants. United States: N. p., 2017. Web.
Bradbury, Andrew M., Kiss, Csaba, & Waldo, Geoffrey S. Method of generating ploynucleotides encoding enhanced folding variants. United States.
Bradbury, Andrew M., Kiss, Csaba, and Waldo, Geoffrey S. Tue . "Method of generating ploynucleotides encoding enhanced folding variants". United States. doi:. https://www.osti.gov/servlets/purl/1354812.
@article{osti_1354812,
title = {Method of generating ploynucleotides encoding enhanced folding variants},
author = {Bradbury, Andrew M. and Kiss, Csaba and Waldo, Geoffrey S.},
abstractNote = {The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue May 02 00:00:00 EDT 2017},
month = {Tue May 02 00:00:00 EDT 2017}
}

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