|Selecting the right detergent for extraction and purification of a membrane protein is often the most challenging and time consuming step. At Anatrace®, we believe in employing a systematic approach to detergent selection, such as our Analytic Extractor assay. But what do you do when the “classic” detergents (DDM, OG, LDAO, C12E8) fail to extract and stabilize your protein, and the right detergent still cannot be found?|
In this constantly evolving field, new reagents and tools are continually being developed to facilitate the extraction and purification of membrane proteins. Anatrace is fully committed to synthesizing these reagents, and our newer detergents, including LMNG and Amphipol A8-35, have been highly successful in the hands of researchers. Another recent addition to the Anatrace product line, glyco-diosgenin (GDN)(1) is also showing great promise, and can be tried in parallel or after the standard detergents and approaches do not work.
GDN is derived from diosgenin and contains a di-maltose hydrophilic group linked to a steroid-based hydrophobic group(1). The CMC of GDN is 18 µM, which is smaller compared to the CMC of DDM (0.17 mM). GDN has been shown to increase the stability of a wide range of membrane proteins when compared to DDM, including:
Bacteriorhodopsin - GDN was shown to be more effective than both DDM and OTG at maintaining the native structure of the protein over a period of 20 days.
Leucine transporter, LeuT - This protein retained activity over 12 days in GDN while activity was lost over this time period in DDM.
Human β2 adrenergic receptor-T4-lysozyme fusion protein (β2AR-T4L) - When exchanged into higher concentrations of GDN, this protein was shown to have a higher thermostability compared to DDM.
Melibiose permease, MelB - A recent study has shown the utility of GDN in increasing the stability of this protein. This increased stability allowed for the detection of galactoside binding by various MelB homologs and mutants, whereas in the detergents DDM and UDM, no binding is observed(2).
Finally, the efficacy of GDN as an extraction detergent was tested using the wild-type β2AR and the δ-opioid receptor (both GPCR proteins). In both cases, GDN solubilized protein showed increased activity compared to the protein solubilized in other detergents.
Researchers have also noted that GDN can be potentially used as a drop-in substitute for the classic detergent, Digitonin. This is most likely attributed to the steroidal basis of both detergents. Digitonin is highly toxic, and since it is extracted and not synthesized, there is the potential for variability in the quality of the detergent. Like GDN, Digitonin has also been successfully used in the extraction of a GPCR protein (M2 muscarinic acetylcholine receptor)(3). Both GDN and Digitonin have similar formula weights; however GDN has a much smaller CMC (18 µM) compared to Digitonin (0.5 mM).
1. Chae, P. S., et al. (2012) Chemistry 18 (31), 9485-9490.
2. Amin, A., et al. (2015) Biochemistry 54 (38), 5849-5855.
3. Haga, K., et al. (2012) Nature 482 (7386), 547-551.