LanFP10-A, first functional fluorescent protein whose chromophore contains the elusive mutation G67A

•First functional autofluorescent protein containing the G67A mutation•Assembly and cloning of two synthetic genes that encode GFP-like proteins•Analysis of GFP-like proteins containing the chromophore-forming tripeptide GYA•Biochemical analysis of GFP-like proteins encoded in Branchiostoma floridae

Since Green Fluorescent Protein (GFP) was first successfully expressed in heterologous systems in 1994, many genes encoding other natural autofluorescent proteins (AFPs) have been cloned and subsequently modified by protein engineering to improve their physicochemical properties. Throughout this twenty-two-year period, glycine 67 (Gly67) has been regarded as the only amino acid in the entire protein family that is essential for the formation of the different reported chromophores. In this work, we demonstrate that a synthetic gene encoding LanFP10-A, a natural protein encoded in the genome of the lancelet Branchiostoma floridae containing the G67A mutation, produces a heterologous, functional yellow fluorescent protein when expressed in E. coli. In contrast to LanFP10-A, LanFP6-A, a second GFP-like protein found in the lancelet genome that also contains the natural G67A mutation, was non-fluorescent.