Extending the Half-Life of a Protein <i>in Vivo</i> by Enzymatic Labeling with Amphiphilic Lipopeptides
Mari Takahara, Shinichi Mochizuki, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Kazuo Sakurai, Noriho Kamiya
Abstract
Synthesis of lipid–protein conjugates is one of the significant techniques in drug delivery systems of proteins; however, the intact conjugation of a lipid and protein is yet challenging due to the hydrophobicity of lipid molecules. In order to facilitate easy handling of the lipid moiety in conjugation, we have focused on a microbial transglutaminase (MTG) that can ligate specific lysine (K) and glutamine (Q) residues in lipopeptides and a protein of interest. As MTG substrates, monolipid- and dilipid-fused amphiphilic short lipopeptide substrates (lipid-G3S-RHK or lipid2-KG3S-RHK) were designed. These amphiphilic lipopeptides and a model protein (enhanced green fluorescent protein, EGFP) fused with LLQG (LQ-EGFP) were both water-soluble, and thus lipid–protein conjugates were efficiently obtained through the MTG reaction with a >80% conversion rate of LQ-EGFP even using cholesterol-G3S-RHK. In vitro cell adhesion and in vivo half-life stability of the successfully obtained lipid–protein conjugates were evaluated, showing that the monocholesterol-G3S-RHK modification of a protein gave the highest cell adhesion efficiency and longest half-life time by formation of a stable albumin/lipid–protein complex.