Abstract

The functional and structural characterization of complex membrane proteins remains a significant challenge in biochemistry and structural biology, largely due to difficulties in achieving high-yield expression of stable, correctly folded constructs. This study addresses this bottleneck by developing a novel platform for the incorporation of **unnatural amino acids (uAAs)** into membrane proteins using the protozoan parasite *Leishmania tarentolae*. The organism's robust machinery for expressing complex eukaryotic proteins, coupled with its ease of genetic manipulation, makes it an ideal host. We engineered a dedicated orthogonal aminoacyl-tRNA synthetase/tRNA pair into *L. tarentolae* to expand its genetic code, enabling site-specific incorporation of various uAAs, including those with biophysical probes or modified side chains for enhanced stability or labeling. Our results demonstrate successful, high-level expression of several challenging multi-pass membrane proteins with incorporated uAAs, confirmed by mass spectrometry and fluorescence spectroscopy. This platform provides a powerful, versatile tool for the *in vivo* study of membrane protein structure, dynamics, and function, significantly advancing our capacity to produce high-quality material for drug discovery and biophysical analysis.