Freestyle f17 expression medium

Example 14

The expression plasmids for transfection were propagated in E. coli and prepared using the EndoFree Mega Kit from Qiagen. Lipocalin muteins were produced by transient transfection of HEK293 cells growing in FreeStyle F17 expression medium from Life Technologies. Cells were cultivated at 37° C. in a Kuehner ISF1-X incubator shaker at 110 rpm with 8% CO₂. Cells were removed six to seven days after transfection by centrifugation for 20 min at 4500 g. Supernatants were filtered via a 0.2 μm membrane to remove remaining particles. His-tagged proteins were purified by affinity chromatography on HisTrap columns from GE Healthcare. To avoid unspecific binding, 20 mM imidazol was added before capture step. The proteins were washed with MES buffer (50 mM MES, 1 M NaCl, pH 6.5) and Tris buffer (50 mM Tris, 50 mM NaCl pH 8) and eluted with a gradient up to 500 mM imidazol in Tris buffer. Polishing step consisted of size exclusion chromatography (SEC) using Superdex 75 (GE Healthcare) with PBS (Life Technologies). After concentration by ultrafiltration, the proteins were sterile filtered (0.2 μm), aliquoted and stored at −80° C. Protein concentration was determined by measurement of absorbance at 280 nm. Further quality control was done by analytical SEC, SDS-PAGE and mass spectrometry.

Example 14

The expression plasmids for transfection were propagated in E. coli and prepared using the Endo Free Mega Kit from Qiagen. Lipocalin muteins were produced by transient transfection of HEK293 cells growing in FreeStyle F17 expression medium from Life Technologies. Cells were cultivated at 37° C. in a Kuehner ISF1-X incubator shaker at 110 rpm with 8% CO₂. Cells were removed six to seven days after transfection by centrifugation for 20 min at 4500 g. Supernatants were filtered via a 0.2 μm membrane to remove remaining particles. His-tagged proteins were purified by affinity chromatography on HisTrap columns from GE Healthcare. To avoid unspecific binding, 20 mM imidazol was added before capture step. The proteins were washed with MES buffer (50 mM MES, 1 M NaCl, pH 6.5) and Tris buffer (50 mM Tris, 50 mM NaCl pH 8) and eluted with a gradient up to 500 mM imidazol in Tris buffer. Polishing step consisted of size exclusion chromatography (SEC) using Superdex 75 (GE Healthcare) with PBS (Life Technologies). After concentration by ultrafiltration, the proteins were sterile filtered (0.2 μm), aliquoted and stored at −80° C. Protein concentration was determined by measurement of absorbance at 280 nm. Further quality control was done by analytical SEC, SDS-PAGE and mass spectrometry.