Native chemical ligation (NCL)¹ has enabled the chemical synthesis of hundreds of modified proteins, however, the generation of larger proteins >180 residues via this methodology is a significant undertaking. Muir and co-workers developed a semi-synthetic variant of NCL, labelled expressed protein ligation (EPL), that enables the generation of larger modified protein targets by fusing a recombinant protein to a synthetic modified peptide fragment.² Unfortunately, EPL typically requires millimolar concentrations of substrates for the reaction to proceed, restricting its applicability for accessing poorly soluble or aggregation-prone proteins. To solve this problem, we recently developed a novel selenium-mediated semi-synthetic methodology, dubbed the expressed protein selenoester ligation (EPSL).³ EPSL offers several key advantages over NCL-based methods, including: 1) the ability to perform ligations at µM-nM concentrations and 2) the ability to chemoselectively deselenize post ligation to preserve native Cys residues in the structure. The power and utility of EPSL will be presented here by highlighting its use in the efficient semi-synthesis of differentially phosphorylated variants of the oligomeric chaperone protein Hsp27 and lipidated analogues of YPT6.