Not Without a Chaperone Vector!

Though recognized as prime drug targets, membrane proteins largely remain terra incognito for structural biologists because they are so difficult to express in eukaryotic-cells. Michael Mendez of Gryffin Consulting, a genetic engineering firm, explains that the main difficulty in expressing membrane proteins is cell toxicity: Overexpressed proteins that cannot be processed in the endoplasmic reticulum (ER) initiate apoptosis, killing the cell. Altering the processing mechanism would prevent cell death and allow the cells to churn out more usable protein.

Enter AB Vector http://www.abvector.com. The San Diego-based company has developed ProFold vectors for co-expression of molecular chaperones with the protein of interest. Molecular chaperones enable proper folding of proteins, preventing them from forming inclusion bodies and rendering them less toxic to cells. Peter Pingerelli, business development manager for AB Vector, says previous efforts to co-express target proteins with chaperones failed largely because they used separate vectors to encode the two genes and could not guarantee that all components were expressed at the required levels. ProFold vectors, on the other hand, contain the genes encoding the chaperone and the target protein on the same construct, ensuring that the proteins and chaperones truly are co-expressed, says Pingerelli.

AB Vector offers two baculovirus-based vector lines: the ER series, for G protein-coupled receptors (GPCRs) and other receptors that must be translocated to the ER and often glycosylated for proper folding; and the C series, for cytosolic and cytoplasmic proteins that do not require translocation. The C Series vectors encode for the cytoplasmic chaperones Hsp40 and Hsp70; the ER series vectors encode for protein disulfide isomerase (PDI), which facilitates disulfide bond formation, and calreticulin, a glycoprotein-folding chaperone. All constructs include a GFP marker to monitor virus propagation.

Mendez, who calls membrane proteins the "next frontier" of the protein-expression industry, is in the early stages of evaluating the technology on GPCRs and Trp channels. Mendez says he has seen an increase in expression over conventional baculovirus vectors and believes this approach to modifying the baculovirus vectors will be the key to membrane protein overexpression.

The ProFold system costs $695 for five transfections. It is compatible with baculovirus systems from BD Biosciences-Clontech and BD Biosciences-Pharmingen. AB Vector also offers custom services using the ProFold technology.

- Aileen Constans