The creation of a novel fluorescent protein by guided consensus engineering.

Consensus engineering has been used to increase the stab-ility of a number of different proteins, either by creatingconsensus proteins from scratch or by modifying existingproteins so that their sequences more closely match a con-sensus sequence. In this paper we describe the ﬁrst appli-cation of consensus engineering to the ab initio creation ofa novel ﬂuorescent protein. This was based on the align-ment of 31 ﬂuorescent proteins with >62% homology tomonomeric Azami green (mAG) protein, and usedthe sequence of mAG to guide amino acid selection at pos-itions of ambiguity. This consensus green protein is extre-mely well expressed, monomeric and ﬂuorescent with redshifted absorption and emission characteristics comparedto mAG. Although slightly less stable than mAG, it isbetter expressed and brighter under the excitation con-ditions typically used in single molecule ﬂuorescence spec-troscopy or confocal microscopy. This study illustrates thepower of consensus engineering to create stable proteinsusing the subtle information embedded in the alignment ofsimilar proteins and shows that the beneﬁts of thisapproach may extend beyond stability.Keywords: ﬂuorescent proteins/consensus engineering/Azamigreen/protein stabilityIntroductionConsensus engineering (Steipe et al., 1994; Steipe, 2004) is anapproach to increase the stability of proteins by modifying aprotein sequence so that it more closely resembles a consensusderived from the alignment of numerous proteins of a particu-lar family. This was initially applied to immunoglobulin vari-able domains (Steipe et al., 1994; Ohage and Steipe, 1999;Wirtz and Steipe, 1999), and subsequently to scFvs (Knappiket al., 2000; Visintin et al., 2002; Arndt et al., 2003), scFvfusions (McDonagh et al., 2003), whole antibodies(Whitcomb, 2003)and isolated CH3 domains (Demarest et al.,2004). This has proved particularly helpful in the use of scFvsas intracellular inhibitors of protein function. As antibodies aresecreted proteins, with a large part of their stability conferredby disulﬁde bonds, the reducing environment of the cytoplasmprevents the formation of such disulﬁde bonds, so diminishingcytoplasmic stability and functionality. scFvs which have beenfound to be naturally stable in the cytoplasm tend to havesequences resembling consensus V-region sequences (Visintinet al., 2002), and the mutation of scFvs to sequences closer tothe consensus signiﬁcantly increases their stability, allowingthem to function under reducing conditions (Ohage and Steipe,1999;Wirtz and Steipe, 1999).The use of this concept has notbeen limited to antibody domains, but has also been applied tothe stabilization of GroEL minichaperones (Nikolova et al.,1998; Wang et al., 1999, 2000), WW (Jiang, 2001) and SH3domains (Maxwell, 1998). Recently, a consensus version ofgp120 was designed and expressed for use as a vaccine (Gaoet al., 2005), and although stability data were not reported, itwas extremely well expressed compared to gp120 derivedfrom single parental isolates.A strikingly successful example of consensus engineeringwas the application to phytases (Lehmann et al., 2000a, 2000b,2001, 2002), enzymes used in animal feed technology. In initialexperiments, a consensus sequence based on 13 sequences wasproduced by gene assembly. This showed an increase in stabilityof 228C over the best parental sequence (Lehmann et al.,2000b), which could be increased an additional 30 8C(to90.48C) by eliminating a number of destabilizing mutations andincluding more sequences in the consensus. The ﬁnal consensussequences differed from the closest parental sequences by 18%(80 residues over 444). Unlike other applications of consensusengineering, which tend to be gradualist, with the introductionof mutations in small numbers, the ﬁrst consensus sequence(Lehmann et al., 2000b) in this study was produced using anab initio approach with the gene synthesized directly. Althougha similar approach was used to generate the V genes used in aphage antibody library containing modular consensus frame-works (Knappik et al., 2000), the consensus sequences usedwere relatively similar to the closest individual V genes, andchanges in stability were not studied.The stabilization of proteins is important for improvedhalf-life and function under the adverse conditions encoun-tered in biotechnological and pharmacological applications.It has also been regarded as important in the use of proteinsas scaffolds to generate libraries of speciﬁc binders. It hasbeen reasoned that if the starting scaffold is more stable, itwill be more tolerant to the destabilizing effects ofmutations, or insertions, used to mediate binding. This wasthe rationale used in the creation of the phage antibodylibrary described above. More recently, it has been applied toankyrins. These are widely distributed, soluble repeat pro-teins generally involved in protein–protein interactions, witheach polypeptide repeat composed of 31 amino acids. A con-sensus sequence created by the alignment of over 2000ankyrins was used as a scaffold for diversity by mutation ofspeciﬁc exposed residues within three repeats (Kohl et al.,2003). The high stability of the consensus ankyrin (denatura-tion requires boiling in urea) explains why artiﬁcial ankyrinsselected from these libraries, with Tms of 65–858C (Binzet al., 2003), are more stable than most other scaffolds.Although consensus engineering has not been applied toﬂuorescent proteins, a recent study used gene synthesis torecreate putative ancestral ﬂuorescent proteins (Ugalde et al.,2004;Chang et al., 2005).Three different evolutionary algor-ithms (Yang, 1997) were used, with amino acids, codons or