Engineering highly stable variants of Corynactis californica green fluorescent proteins

Abstract

Fluorescent proteins are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered fluorescent proteins such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split fluorescent protein from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to three‐fold faster complementation than split GFP. We present the biophysical, biochemical and structural characteristics of 5 full length variants derived from these split Corynactis californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.This article is protected by copyright. All rights reserved.