Post-translational modifications (PTMs) are fundamental to many biological processes and contribute enormously to the diversity of protein structure and function. Lipidation of bacterial proteins is a highly conserved process, whereby proteins possessing a lipobox motif are di- and triacylated at an N-terminal cysteine residue.^1,2 These lipoproteins are potent agonists of the innate immune system through toll-like receptor 2 (TLR-2).³ Existing methods to study these PTMs are inadequate; for example, recombinant expression leads to a heterogeneous mixture of PTMs, meaning it is difficult to elucidate their functional effects with any level of precision. Protein semisynthesis, whereby larger and unmodified recombinant protein fragments are ligated to smaller synthetic and modified fragments, is a powerful approach for generating larger modified proteins.⁴ To date, native chemical ligation has been used successfully for a large number of modified proteins, however, is not sufficient for proteins with poor solubility (such as lipoproteins) due to the high fragment concentrations required for the reaction to proceed. We have recently developed a novel semisynthetic approach for the production of mycobacterial lipoproteins bearing di- and triacylated N-terminal cysteines which will be described in this presentation.