Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin

The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin- like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from E. coli extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex E. coli extracts by repeated precipitation/re-dissolution steps at near physiological temperature. • The method is based on the inverse thermal transition process. • The “green ”method is affordable for basic laboratories and can be easily transferred to new users.