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The FP eCGP123 was engineered using directed evolution from a rationally designed and relatively unstable FP, consensus green protein (CGP)24. Evolution of eCGP123 involved a recursive process whereby a destabilizing amino acid sequence (based on an antibody binding loop) was inserted at pre-defined positions between strands in the β-barrel leading to loss of fluorescence, followed by mutagenesis of the rest of the scaffold to recover fluorescence13. As beneficial mutations accumulated through DNA shuffling, the stringency (number of loops inserted simultaneously) was increased until a pool of FP-encoding genes containing three loops and accumulated stabilizing mutations was generated. When the destabilizing loops were excluded and consensus mutations were combined and included in a synthesized eCGP123 gene, the resulting protein exhibited exceptional thermal and chemical stability13. In this work, we describe the X-ray crystal structure of eCGP123 and report the use of structure-guided engineering to generate a new protein, Thermal Green fluorescent Protein (TGP), with improved solubility and stability compared to eCGP123.
A DNA expression cassette encoding TGP was synthesized, expressed in, and purified from Escherichia coli (see Materials and Methods) in parallel with eCGP123. Improvements in protein solubility were immediately apparent. TGP and eCGP123 protein purified by Ni-NTA chromatography was adjusted to a similar protein concentration (~1.3 mg/ml) and dialyzed against PBS. After overnight equilibration, eCGP123 was clearly aggregated while TGP showed no signs of visible aggregation (Figure 3A–B). It was necessary to increase the concentration of TGP to >50mg/ml before any aggregation became visible. These observations demonstrate that our structure guided surface engineering approach resulted in a protein with greatly improved solubility.
(2020). Communications Biology, 3(1) , . doi: 10.1038/s42003-020-01478-z. Article