The cell-free protein biosynthesis - applications and analysis of the system

The in vitro protein biosynthesis has the potentials to become a powerful t echnology for biochemical research. Beside the determination of structure a nd function the in vitro evolution of proteins is also of great interest. The system described was used to produce bovine heart fatty acid binding pr otein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag II affinity peptide. The proteins were pur ified after and during protein biosynthesis by using a StrepTactin Sepharos e matrix. No significant influence of the Strep-tag and the conditions duri ng the affinity chromatography on maturation or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display . The selection of a specific mRNA coding for a shortened FABP with a N-ter minal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. After nine cycles of transcription, translation, affinity selection and RT-PCR t he protein with the His-tag could be enriched 10(8)-fold. In order to correlate a possible relationship between changes in protein po pulation and biological function studies were initiated in which 2-dimensio nal protein patterns of the total in vitro system were compared after 0 and 2 h reaction time, The very interesting findings are that a number of prot eins disappear, while others are newly formed during protein synthesis.