Intrabodies with defined framework that is stable in a reducing environment and applications thereof
View Patent ↗A method for the isolation of CDRs in a defined framework that is stable and soluble in reducing environment is described as well as thus obtainable scFv. Starting from such scFv with defined framework a scFv library can be generated wherein the framework is conserved while at least one complementary determining region (CDR) is randomized. Such library, e.g. in yeast cells, is suitable for screening for antibody/CDR-interactions or for screening for antibodies.
1. A method for the identification of intrabody frameworks or intrabodies which are soluble and stable in reducing conditions comprising the following steps:
transformation of suitable host cells a nucleic acid library, said library encoding a fusion product comprising an intrabody and a transcriptional activation domain wherein said transcriptional activation domain is only active as part of a fusion protein comprising an intrabody moiety which is soluble and stable and
culturing said cells under conditions allowing the identification and selection of cells expressing an intrabody moiety which is soluble and stable in reducing conditions by detection of a reporter gene that is expressed by the interaction of the transcriptional activation domain with a DNA binding domain in the host cell wherein the interaction of the transcriptional activation domain with the DNA binding domain results in the growth or identification of cells indicating that the intrabody frameworks or intrabodies are stable and soluble under reducing conditions and is not dependent upon the presence of the antigen for which the intrabody is specific.
2. The method of claim 1 , wherein said reporter gene expresses a selectable activity.
3. The method of claim 2 , wherein said reporter gene expresses an enzymatic activity or fluorescence activity.
4. A method for the identification of intrabody frameworks or intrabodies which are soluble and stable under reducing conditions comprising:
transformation of suitable host cells harboring a nucleic acid library said library encoding a fusion protein comprising an intrabody and a DNA binding protein that can activate transcription and said cells further comprise a reporter gene encoding a detectable protein, said marker gene being under transcriptional control of said DNA binding protein wherein the activation of transcription is not dependent upon the presence of an antigen for which the intrabody is specific, and
cultivation of said cells under conditions allowing the identification and selection of cells expressing a fusion protein comprising a soluble and stable intrabody in the selected conditions by detection of the protein encoded by said reporter gene.
5. A method for the identification of intrabody frameworks or intrabodies which are soluble and stable under reducing in selected conditions comprising:
transformation of host cells with a DNA encoding a first protein comprising an intrabody and one part of a transactivation system which is a transcriptional activation domain wherein said transcriptional activation domain is only active as part of a fusion protein comprising an intrabody moiety which is soluble and stable and
said cells further-express a second protein comprising at least the second part of said transactivation system which is a DNA binding domain wherein the interaction of the transcriptional activation domain with a DNA binding domain in the host cell results in the growth or identification of cells indicating that the intrabody frameworks or intrabodies are stable and soluble under reducing conditions and is not dependent upon the presence of the antigen for which the intrabody is specific, whereby said transactivation system is linked to a survival allowing marker gene which is under transcriptional control of said transactivation system and
identifying cells expressing a first and a second protein interacting with each other via a constant region of the first protein by selecting for expression of said marker gene in the selected conditions.
6. The method of claim 5 , wherein said first library encoded proteins comprises a transcriptional activation domain and said second proteins comprises a DNA binding domain or said first library encoded proteins comprises a DNA binding domain and said second proteins comprises a transcriptional activation domain.
7. The method of claim 5 , wherein said second proteins comprises a DNA binding domain or a transactivation domain, respectively, and a protein interacting with a constant region of said first library encoded protein.
8. The method of claim 5 wherein said first library encoded protein comprises the transcription activation domain of GAL4 and Gal11P and said second protein comprises the DNA binding domain of Gal4.
9. The method of claim 1 , wherein the host cell is an eukaryotic cell.
10. The method of claim 9 , wherein the eukaryotic cell is a yeast cell.