We used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA- synthetase:suppressor tRNA pair in Escherichia coli, to directly insert the non- canonical amino acid 5-OH tryptophan (5-OHTrp) at position 72 in human apoA-I. Characterization of recombinant human apoA-I by mass spectrometry confirmed successful site-specific incorporation of Trp(5-OH) in apoA-I.