Efficient purification of human transmembrane protein, mutant hMC4R TM2.

The human melanocortin‐4 receptor (hMC4R) is a receptor of the alpha‐melanocyte stimulating hormone (α‐MSH) which regulates food intake and energy consumption. It is therefore considered to be a main part of human energy homeostasis and body weight. It has been reported that a D90N mutation in the second transmembrane domain (TM2) was especially found in the patients with severe‐early onset obesity. We expressed the mutant types of hMC4R TM2 (m‐hMC4R TM2) to consider the relationship between the function and the structure. When the expressed m‐hMC4R TM2 was purified, sodium dodecyl sulfate (SDS) was used as a detergent in a fast protein liquid chromatography (FPLC) system to separate the protein based on its hydrophobicity. However, SDS binds too strongly to m‐hMC4R TM2, interfering with the various techniques used for structural characterization. Thus, we discovered more efficient SDS removal methods by using KCl‐SDS precipitation, resulting in improved resolution and sensitivity of the analytical techniques. The high yield of m‐hMC4R TM2 obtained from the diverse optimizations was confirmed and characterized by various techniques such as CD, MALDI‐TOF MS, and solution NMR spectroscopy. [ABSTRACT FROM AUTHOR]