Cytochrome b5 is an electron transport protein found in bacteria, protozoans, yeasts and mammals. This membrane protein is required for the activity of stearoyl-CoA desaturase (SCD), an integral membrane desaturase that plays a role in obesity, diabetes, hypertension, cardiovascular disease, immune disorders, degenerative neurological diseases and skin diseases.

Cytochrome b5 is a useful tool for researchers studying SCD activity. However, current methods of making large quantities of functional cytochrome b5 to use in assays are labor intensive and time consuming. Improved techniques for synthesizing full length cytochrome b5 are needed.

UW-Madison researchers have developed a simpler method of producing and purifying functional membrane polypeptides, such as cytochrome b5. This method utilizes a recombinant expression vector that expresses a fusion of cytochrome b5 and a solubilizing agent, such as maltose binding protein (MBP).

The vector can be used to express the fusion protein in solution. The protein optionally can be purified, and then a simple chemical or enzymatic cleavage reaction can release the MBP moiety when the functional, full length cytochrome b5 is needed.

In addition, the inventors discovered that the carboxy terminus of the cytochrome b5 peptide functions as a membrane anchor. To use this method to synthesize other functional membrane proteins that do not have a C-terminal membrane anchor, this anchor section can be fused to the protein, allowing it to spontaneously attach to a membrane when the solubilizing agent is released.

File Number: P07407US 

Other Information: For an assay that uses the cytochrome b5 synthesized using this technology, see WARF reference number P06127US.