The creation of a novel fluorescent protein by guided consensus engineering

Abstract

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.

Advances in fluorescent protein technology

“…The construction of new FP variants using guided consensus sequence engineering has been applied (Dai et al, 2007) to a monomeric green variant, Azami Green (mAG), derived from the stony coral genus Galaxeidae (Karasawa et al, 2003). Consensus engineering attempts to alter protein properties by modifying the sequence to one that more closely resembles a consensus derived from a large population of similar proteins in a particular family.…”

Simple Preparation of Green Fluorescent Protein Conjugated with β-Cyclodextrin in a Site Specific Manner

“…1). This strategy may also be expanded to similar fluorescent proteins [31][32][33] and β-CD like molecules for grafting. Fig.…”

GFP-Based Biosensors

“…The resulting protein CGP (consensus green protein) has comparable expression to the parent protein with increased brightness and slightly decreased stability [32]. A novel directed evolution process was then carried out on CGP to stabilize it by inserting destabilizing loops into the protein, then evolving it to tolerate the insertions, then removing the destabilizing loops.…”