Title: Method For Determining And Modifying Protein/Peptide Solubilty

Abstract

A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

Inventors:

Waldo, Geoffrey S ^[1]

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1. Espanola, NM

Issue Date:

2005-03-15

Research Org.:

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

OSTI Identifier:

879816

Patent Number(s):

6867042

Application Number:

10/217995

Assignee:

The Regents of the University of California (Los Alamos, NM)

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}
C07K14/47 - from mammals
C07K2319/02 - containing a signal sequence
C07K2319/60 - containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
C07K2319/61 - containing an enzyme fusion for detection

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/62 - DNA sequences coding for fusion proteins

C - CHEMISTRY C12 - BIOCHEMISTRY C12P - FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {

C12P21/02 - having a known sequence of two or more amino acids, e.g. glutathione

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6897 - involving reporter genes operably linked to promoters

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

G01N2800/52 - Predicting or monitoring the response to treatment
G01N33/68 - involving proteins, peptides or amino acids {

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DOE Contract Number:  

W-7405-ENG-36

Resource Type:

Patent

Country of Publication:

United States

Language:

English