Use of microcompartmentation of transcription/translation complexes in emulsions in the selection of proteins with novel properties
An in vitro method of assocg. proteins with the nucleic acid encoding them in the selection of novel variants is described. This method can be used for evolutionary selection of polypeptides in vitro. The inventive method makes it possible to allocate nucleic acids to the polypeptides coded thereby and to select and isolate nucleic acids coding for polypeptides with selected properties. The method involves manuf. of the protein of interest in vitro in a coupled transcription-translation system. The protein is manufd. as a fusion protein with a nucleic acid-binding protein that will bind to the nucleic acid upon appearance of the translation product. The transcription-translation systems are compartmented by incorporating them into a water-in-oil emulsion. The nucleic acid-binding protein is preferably a (cytosine-5)-Me transferase. This binds to DNA, and by use of a suicide inhibitor base analog, such as 5-fluorocytosine, incorporated into the DNA, a covalent complex can be formed. Sites contg. the label lie outside the gene for the fusion protein. The complexes can then be captured, e.g. using an affinity label, and screened for the desired property and the gene recovered for further processing. The use of the method to manuf. fusion proteins of HaeIII methylase labeled with hexahistidine and FLAG peptides is demonstrated. Use of calmodulin as an affinity label is also demonstrated. [on SciFinder (R)]
34383955
CAN 142:330803
3-1
Biochemical Genetics
Patent
written in German.
20050407
848779-26-0 Role: BUU (Biological use, unclassified), PRP (Properties), BIOL (Biological study), USES (Uses) (as suicide substrate for restriction methyltransferase; use of microcompartmentation of transcription/translation complexes in emulsions in selection of proteins with novel properties); 10356-76-0D (5-Fluorodeoxycytidine) Role: BUU (Biological use, unclassified), BIOL (Biological study), USES (Uses) (as suicide substrate; use of microcompartmentation of transcription/translation complexes in emulsions in selection of proteins with novel properties); 84177-56-0DP; 85537-81-1DP (DNA methyltransferase MspI); 91448-96-3DP (HhaI methyltransferase); 92228-38-1DP (DNA methyltransferase AluI); 92228-40-5DP (HaeIII methyltransferase); 149719-23-3DP Role: BPN (Biosynthetic preparation), BUU (Biological use, unclassified), BIOL (Biological study), PREP (Preparation), USES (Uses) (use of microcompartmentation of transcription/translation complexes in emulsions in selection of proteins with novel properties); 1338-43-8 (Span 80); 9005-65-6 (Tween 80) Role: BUU (Biological use, unclassified), BIOL (Biological study), USES (Uses) (water-in-oil emulsions contg.; use of microcompartmentation of transcription/translation complexes in emulsions in selection of proteins with novel properties)
A1
CH
2003-1671
20031001
Alternative title(s) : (de) Verfahren zur in vitro evolution von polypeptiden (fr) Procede pour faire evoluer des polypeptides in vitro (en) Method for in vitro evolution of polypeptides
Application: WO WO, (ETH Zuerich, Switz.).
| Identifier | Country code | Kind code | Date issued |
JP4430076 | JP | B2 | 2010-03-10 |
US7527954 | US | B2 | 2009-05-05 |
ES2300808 | ES | T3 | 2008-06-16 |
DE502004006204 | DE | D1 | 2008-03-27 |
AT386113 | AT | T | 2008-03-15 |
EP1668126 | EP | B1 | 2008-02-13 |
US2007105117 | US | A1 | 2007-05-10 |
JP2007507210 | JP | A | 2007-03-29 |
EP1668126 | EP | A1 | 2006-06-14 |
WO2005030957 | WO | A1 | 2005-04-07 |