Insect cells

The initial steps in the cloning procedure are the same as for the bacterial system. We are using the LIC-technology as described in Chapter 19 of Baculovirus Expression Vector System: An Emerging Host for High-Throughput Eukaryotic Protein Expression (Methods in Molecular Biology, Vol 439: Genomic Protocols: Second Edition).

Most of the targets are fused with an N-terminal hexahistidine and a TEV-protease cleavage site for optional tag removal. Proteins targeted for secretion are fused with an additional signal peptide (from baculovirus gp64). The main vectors in use are pFB-LIC-Bse (GenBank accession EF199842) and pFB-Sec-NH (Plasmid 39189).

We are using the Bac-to-Bac system (Invitrogen) modified according to Methods in Molecular Biology (mentioned above). DH10Bac cells are used for recombinant bacmid production and blue/white colony screening is performed to confirm the insertion of the target gene. Sf9 or High Five insect cells are then transfected with the bacmid and recombinant viruses are produced.

Cultures for P1 virus generation are used for the small-scale screening. In short, we use a 24-deep well plate to grow all cultures in a volume of 3 mL Sf-900 II media. The cells are harvested after 48-72 h and we take out a small sample from the liquid culture and verify the presence of target protein using SDS-PAGE. We also continue to perform cell lysis, clarification and micro-scale IMAC to assess the levels of soluble and purifiable target protein from each clone. The purification of membrane proteins is performed in the presence of 1% FC12.

Clones that score sufficiently high in the small-scale expression analysis will be subjected to large scale expression.

P2 virus generation is mostly used for the large-scale expression (expression volumes varying from 1-5 L). The purification and downstream process largely follow the protocols used in E. coli protein production.