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:

Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.

Issue Date:

2017-05-02

Research Org.:

Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1354812

Patent Number(s):

9637528

Application Number:

12/286,967

Assignee:

Los Alamos National Security, LLC

Patent Classifications (CPCs):

C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07K - PEPTIDES

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

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C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07K - PEPTIDES

C07K14/43595 - {from coelenteratae, e.g. medusae}
C07K16/00 - Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies {
C07K2317/565 - Complementarity determining region [CDR]

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/1058 - {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}

G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N33/533 - with fluorescent label
G01N33/582 - {with fluorescent label}

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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