Biosynthetic Production of an isotopically Labelled Retinal in E. Coli

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging biophysical technique which has been useful in studying the structure of integral membrane proteins such as microbial rhodopsins. However, this technique requires the incorporation of isotopically-labelled atoms into the protein. This is usually accomplished through over-expression of rhodopsins in E. coli, in minimal media wherein all carbon and nitrogen sources are isotopically labeled. The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, the retinal binding pocket is of significant interest for ssNMR assignments. Unfortunately, the de novo organic synthesis of an isotopically-labelled retinal is cost-prohibitive in large scale expression. Previously, the biosynthesis of a retinal precursor, beta-carotene, has been introduced into many different organisms. This system can be extended to the E. coli expression strains UT5600 and BL21. We have shown that the novel biosynthetic production of an isotopically labelled retinal ligand concurrently with its apoprotein proteorhodopsin in E. coli presents a cost effective alternative to de novo organic synthesis. By using alternately labeled carbon sources (glycerol) we were able to verify the biosynthetic pathway for retinal and assign several new carbon resonances for proteorhodopsin-bound retinal.