Harnessing Split‐Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Abstract

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogon-ality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair Ava N /Npu C can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.

Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels

“…In some cases, these issues can be overcome by alternative approaches such as protein semisynthesis using protein trans-splicing (PTS). [21][22][23][24] PTS involves formation of a peptide bond between two proteins or protein fragments (termed as exteins) by means of traceless native chemical ligation mediated by a split intein.…”