Expression in HEK293E cells. (A) pTT5-eGFP and pTT22-CD40L (GenBank NT_039706) constructs were generated by restriction enzyme cloning. pCoofy40L-eGFP was generated using standard LP1 (3C) and LP2 (ccdB) primers. Coofy40-CD40L was generated using pTT22-CD40L as template for PCR amplification of the insert. Gene specific LP1 primer sequence included vector VEGF signal sequence from pTT22. Recombinant plasmids were transfected into HEK293E cells and expressed for 72h. Total lysates of untransfected cells, pTT5-eGFP (lane 1) pCoofy40-eGFP (lane 2), cell culture supernatants of untransfected cells, pTT5- CD40L (lane 3) and pCoofy40-CD40L (lane 4) detected by Coomassie, silver staining and Western Blot HisProbe™-HRP detection. (B) eGFP was SLIC cloned into pTT derivative Coofy40 using standard LP1 (3C) primer in combination with either His10, OneStrep, S, CBP, HPC4 LP2 primer to introduce a C-terminal tag. Recombinant plasmids were transfected into HEK293E cells end expressed for 72h. Total lysates of untransfected cells, pCoofy40-eGFP fused to C-terminal S-tag (lane 1), CBP (lane 2), HPC (lane 3), OneStrep (lane 4) and His10 (lane 5). Eluates from S-Protein Agarose (lane 6), Calmodulin Affinity resin (lane 7), His-Select washed with 50 column volumes (CV) 20mM imidazole buffer (lane 8), His-Select washed with 50 CV 20mM imidazole buffer, 20 CV 50mM imidazole buffer and 20 CV 80mM imidazole buffer (lane 9), Calmodulin Affinity resin (lane 10, same sample as lane 7), Anti-Protein C Affinity matrix (lane 11) and Strep-Trap™ (lane 12). Yields estimated for His10 and OneStrep based on Bradford staining was 8 mg / L HEK culture and 3 mg /L HEK culture respectively.