| abstract |
The present invention relates to the production of variants of a protein in an in vitro evolution method, comprising the steps: (A) provision of an in vitro expression system comprising (i) a nucleic acid sequence S which codes for a protein Y which is to be varied, (ii) a target molecule X which is able to bind to the protein Y and/or at least one variant Y′ thereof, (iii) an RNA polymerase (Pol) which is able to transcribe the nucleic acid sequence S, (iv) a reverse transcriptase (RT) which is capable of reverse transcription of transcripts of the nucleic acid sequence S, where either the target molecule X is coupled to Pol and the protein Y is coupled to RT, or the target molecule X is coupled to RT and the protein Y is coupled to Pol, (B)incubation of the in vitro expression system from (A) under conditions which enable transcription, reverse transcription and translation to form variants Y′ of the protein Y and nucleic acid sequences S′ coding therefor, and which favor the formation of variants Y′ with improved binding properties for the target molecule X, (C) isolation and, where appropriate, characterization of those variants Y′ which exhibit improved binding properties for binding to X, and/or isolation of nucleic acid sequence variants S′ coding for Y′. |