WO2009116984A2 - Highly thermostable fluorescent proteins - Google Patents

Highly thermostable fluorescent proteins Download PDF


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WO2009116984A2 PCT/US2008/013900 US2008013900W WO2009116984A2 WO 2009116984 A2 WO2009116984 A2 WO 2009116984A2 US 2008013900 W US2008013900 W US 2008013900W WO 2009116984 A2 WO2009116984 A2 WO 2009116984A2
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WO2009116984A3 (en
Andrew M. Bradbury
Geoffrey S. Waldo
Csaba Kiss
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Los Alamos National Security, Llc
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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae


Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99 °C for short periods of time without denaturing, and retains 85 % of its fluorescence when heated to 80 °C for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.




This patent application claims the benefit of the filing date of United States Provisional Patent Application No. 61/008,689 filed 12/21/2007.


This invention was made with government support under Contract No. DE- AC52-06 NA 25396 awarded by the U.S. Department of Energy. The government has certain rights in the invention.


GFP and its numerous related fluorescent proteins are now in widespread use as protein tagging agents (for review, see Verkhusha et al., 2003, GFP-like fluorescent proteins and chromoproteins of the class Anthozoa. In: Protein Structures: Kaleidescope of Structural Properties and Functions, Ch. 18, pp. 405-439, Research Signpost, Kerala, India). GFP-like proteins are an expanding family of homologous, 25-30 kDa polypeptides sharing a conserved 1 1 beta-strand "barrel" structure. The GFP-like protein family currently comprises well over 100 members, cloned from various Anthozoa and Hydrozoa species, and includes red, yellow and green fluorescent proteins and a variety of non-fluorescent chromoproteins. A wide variety of fluorescent protein labeling assays and kits are commercially available, encompassing a broad spectrum of GFP spectral variants and GFP-like fluorescent proteins, including DsRed and other red fluorescent proteins (Clontech, Palo Alto, CA; Amersham, Piscataway, NJ.). However, the stability of fluorescent proteins is limited. Various approaches aimed at stabilizing fluorescent proteins have been undertaken. For example, Siemering et al. described the generation of a GFP mutant (GFPA) using site- directed mutagenesis, reporting that the mutant showed reduced sensitivity to temperature in both bacteria and yeast cultured at 37°C (Siemering et al., 1996, Curr Biol 6: 1653). United States Patent No. 6,414,119 described a GFP mutant showing modest improvements in thermal stability over wild type GFP (reportedly retaining fluorescence and solubility at 420C, and showing some fluorescence at 500C). More recently, Pedelacq et al.,7 used directed evolution to increase the stability of GFP by selecting for resistance to the destabilizing effects of a poorly folding and aggregating ferritin sequence fused upstream. The first fusions were very weakly fluorescent, but with further evolution of the GFP, this external destabilization could be overcome and a variant (termed "superfolder GFP") able to resist the folding interference of ferritin was selected. This was shown to be considerably more stable than standard GFP by a number of different measures, including resistance to thermal and chemical denaturation.

A number of different methods have been developed to create thermostable proteins, most of which involve the creation of libraries and the identification of improved proteins by selection or screening. Conceptually, the most straightforward way to identify proteins with improved thermostability has been to apply a thermal challenge to a collection of individual clones and test the remaining functionality of the clones, repeating this process if necessary, to combine useful mutations8"10. A similar method, which does not rely on such extensive screening requirements, involves direct selection of clones growing at elevated temperature within thermophilic bacteria. However, to date, this method has only been applied to the selection of thermophilic antibiotic resistance proteins11' 12, and as laboratory organisms typically do not grow at elevated temperatures, it has been difficult to generalize. As a result, considerable effort has been put into the development of alternative approaches which involve selection or screening for biophysical or biological properties which can serve as surrogates for, and are often correlated with, thermostability.

One of the first examples of this approach is the PROSIDE (protein stability increased by directed evolution)13"20 approach in which resistance to protease digestion is used as the surrogate property for protein stability, with filamentous phage infectivity being the selection modality. Proteins under test are expressed between two domains in g3p (the phage receptor for bacteria): if they are cleaved by protease, the filamentous phage loses the N terminal g3p domain and consequently its ability to infect; if the protein is protease resistant infectivity is maintained. This has been successfully used to increase the stability of the betai domain of protein G15, the cold shock protein of B. subtilis17 and ribonuclease T113. In another approach involving directed evolution, Shusta et al., showed that the display levels of heterologous proteins on the surface of yeast correlated with expression levels and thermal stability21, although exceptions to this have been recently described22.

Consensus engineering23' 24 is an approach to increase protein stability which does not use directed evolution, but the informational content of aligned sequences. By modifying a sequence so that it more closely resembles a consensus derived from the alignment of numerous proteins of a particular family, it has been found that significant increases in stability can be obtained. This has been applied to antibodies and antibody fragments5' 24~31, GroEL minichaperones32' 33, p5334, WW35 and SH3 domains36. More recently consensus engineering has been applied to the creation of novel proteins, rather than the stepwise modification of pre-existing ones to resemble a consensus. Perhaps the most striking success was the application to phytases37"*0, in which a final protein with a Tm of 90.40C was obtained: 52°C greater than the best component parental sequence40. Similar stability was obtained with a consensus ankyrin sequence based on the alignment of 2000 different ankyrins41 "43. We recently applied this method to the creation of a consensus green protein (CGP)44. Although we obtained a functional fluorescent protein, its Tm was 5°C less than the monomeric Azami Green45 used to identify the sequences comprising the consensus. However, in this case no effort was made to examine the effects of individual mutations, and it is likely that some of the consensus mutations were destabilizing, as had been previously shown for the phytase37"40.

Other methods used to increase protein stability, relying heavily on structural information, include "helix capping"46"49 or optimization50"52, the introduction of salt bridges or their replacement by hydrophobic interactions53"59, the introduction of clusters of aromatic-aromatic interactions60"62 and rigidification strategies, in which disulfide bonds or glycine to alanine, or Xaa to proline changes are introduced63"65. However, most of these have been carried out on model structures, and none has been widely adopted.

Thermostabilization of proteins is regarded as important in a number of biotechnological and pharmaceutical applications. Within the context of industrial enzymes, thermostability leads to longer enzyme survival times, as well as more efficient reactions at higher temperatures and diminished microbial contamination, all of which result in diminished costs, while in the pharmaceutical arena, thermostability of protein therapeutics leads to longer half lives and more effective drugs1"3. Thermostability has also been regarded as important in the use of proteins as scaffolds to generate libraries of specific binders. It has been reasoned that if a starting scaffold is more stable, it will be more tolerant to the destabilizing effects of mutations, or insertions, used to mediate binding. This has been shown for affinity reagents based on ankyrins4, and has also been applied to the creation of phage antibody libraries5. Finally, proteins of increased thermostability are more resistant to mutations than the protein from which they are derived, promoting evolvability by providing greater permissivity to mutations leading to novel functions6' 7.


The invention relates novel and highly thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules of the invention. Exemplary TSFPs are provided. In particular, polypeptides comprising eCGPs of the invention, including but not limited to those having the sequences of SEQ ID NOS: 9 and 10, are provided. Additionally, nucleic acid molecules comprising a polynucleotide encoding such polypeptides are also provided, and include without limitation, nucleic acid molecules which comprise polynucleotides encoding the sequences of SEQ ID NOS: 4 and 5. Vectors comprising such nucleic acid molecules are also provided, as are cells comprising such vectors.

The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins. The method of the invention is described, infra, and in the Examples which follow. Briefly, in a simplified description, the method entails internally destabilizing the protein using a heterologous insertion, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization, and then removing the heterologous insert.

The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99°C for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 800C for several minutes.


FIG. 1. Schematic outline of the internal destabilization methodology used to generate thermostable variants of CGP, an artificial fluorescent protein (Dai et al., 2007, supra). (A) strategy applied to CGP. After the insertion of a single insert, CGP loses fluorescence which was regained by mutation and selection. This process was repeated until fluorescent proteins resistant to the destabilizing effects of three loops were obtained. For each evolved fluorescent variant, a gene is synthesized which lacks the inserted loop(s). (B) shows induced bacterial colonies, with a colony expressing CGP before (colony 1) and after insertion of the HCDR3 in loop 1 (colony 2) or loop 2 (colony 3). The remaining colonies are mutated variants, showing the improvement in fluorescence.

FIG. 2. Accumulation of amino acid mutations through iterative internal destabilization. The original sequence of CGP is represented by the small squares at the top, with the positions of amino acids that underwent mutation indicated as black boxes. The exact positions and wild type sequences of these are shown below, with the three insertion sites indicated as inverted red triangles flanked by the exact positions. The mutations occurring at each site, for each evolutionary round and loop insertion strategy, are shown. White squares indicate wild type sequence. Where a mutation has occurred, the letter indicates the new mutation, and the number the percentage of the sequenced fluorescent clones that contain that mutation. This is also represented graphically by that portion of the white square colored green. For example, at position 7, in all early evolutionary rounds 100% of clones changed the wild type aspartate to a glutamate. If more than one mutation is found at a particular site, both amino acids are given with their percentages, indicated by green and yellow boxes. After three rounds when loops 2 and 3 were targeted, 12% of clones also showed a valine at this position, which increased to 50% in later rounds. The percentage of clones carrying a particular mutation are shown if that mutation comprises more than 5% of clones in any of the evolutionary rounds.

FIG. 3. Sequence alignments of various TSFPs of the invention, compared to the reference protein, CGP, and to the protein from which CGP was initially derived, mAG (BAD52002).

FIG. 4. Absorption and emission of purified TSFPs. (A) shows purification and expression levels of the different purified proteins. The amounts given correspond to the total amount of purified protein from 60 ml fermentation volume. (B) shows absorption and emission of the purified CGP, various eCGP proteins, and mAG normalized to 1 for the respective peaks. Peak values are provided in TABLE I.

FIG. 5. Thermal stability of evolved fluorescent proteins. (A) Fluorescence profile of the different proteins gradually heated to 990C and then allowed to recover at 300C. Fluorescence was measured every six seconds, and normalized to the fluorescence level at 3O0C. (B) Enlargement of fluorescence profile from 90-990C, showing the persistence of low levels of fluorescence with eCGP123 and eCGP23 at 99°C. (C) Stability with repeated heating and cooling cycles. Proteins were heated to 99°C for one minute and then cooled to 30°C for two minutes. This was carried out sixty times and fluorescence was measured at the end of each heating or cooling period. (D) The survival of fluorescent proteins at 800C was assessed by heating to 800C, measuring fluorescence every six seconds. Fluorescence was normalized to the fluorescence level after five minutes at 80°C, at which time the initial rapid loss of fluorescence due to heating stabilized. (E) The survival of fluorescent proteins at 80°C was assessed by heating to 800C, and measuring fluorescence each six seconds. Fluorescence was normalized to the fluorescence level after five minutes at 80°C" (FIG. 5D). (F): As FIG. 5E, except proteins were heated to 85°C.

FIG. 6. Stability to chemical denaturation. (A): Each of the fluorescent proteins was diluted into 48 different Guanidium hydrochloride concentrations, with 7.4 M being the highest concentration. The residual fluorescence was measured at equilibrium, normalized and plotted. The recovered fluorescence was normalized by dividing the fluorescence of corresponding non-denatured samples diluted in parallel. (B): Dependence of the standard free energy of denaturation on guanidine concentration assuming a two-state folding model for the fluorescent proteins (TABLE III). (C): Refolding kinetics. Long-term (2000 s) progress curves for recovery of fluorescence during refolding of Gdn HCI-denatured eCGP123 (blue), CGP (magenta), and mAG (green) upon 20- fold dilution of denatured samples in fresh buffer containing 1 mM DTT at 25°C (see Methods), with the inset showing the short-term progress curves. Initial rates V, were obtained from slope at t = 0 s of 2nd-order polynomials fitted to the first 12 s of short-term progress curves. Fluorescence normalized by dividing by final fluorescence value at 15 h.


Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form. Unless specifically limited, the term "polynucleotide" encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 , Nucleic Acid Res. 19: 5081 ; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, MoI. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) lsoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et a/., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. For example, one type of vector is a plasmid, a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors".

The term "host cell" (or "recombinant host cell"), as used herein, refers to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector, and includes not only the particular subject cell but also the progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "link" as used herein refers to a physical linkage as well as linkage that occurs by virtue of co-existence within a biological particle, e.g., phage, bacteria, yeast or other eukaryotic cell.

"Physical linkage" refers to any method known in the art for functionally connecting two molecules (which are termed "physically linked"), including without limitation, recombinant fusion with or without intervening domains, intein-mediated fusion, non-covalent association, covalent bonding (e.g., disulfide bonding and other covalent bonding), hydrogen bonding; electrostatic bonding; and conformational bonding, e.g., antibody-antigen, and biotin-avidin associations.

"Fused" refers to linkage by covalent bonding.

As used herein, "linker" or "spacer" refers to a molecule or group of molecules that connects two molecules, such as VH and VL genes or polypeptides (i.e., in a scFv), and serves to place the two molecules in a preferred configuration.

The term "isolated" refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term "isolated" is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The terms "label" and "detectable label" refer to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" or "detectably labeled" antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. A great number of such labels are known in the art, including without limitation protein tags, radioisotopes, metal chelators, enzymes, fluorescent compounds (dyes, proteins, chemicals), bioluminescent compounds, and chemiluminescent compounds.

The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a fluorescent protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 22 amino acids or nucleotides in length, or more preferably over a region that is 30, 40, or 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981 , Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. MoI. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT1 FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. MoI. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. ScL USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.

The term "as determined by maximal correspondence" in the context of referring to a reference SEQ ID NO means that a sequence is maximally aligned with the reference SEQ ID NO over the length of the reference sequence using an algorithm such as BLAST set to the default parameters. Such a determination is easily made by one of skill in the art.

The invention provides novel and highly stable fluorescent proteins. Because the proteins of the invention are particularly stable at very high temperatures, they have been termed Thermostable Fluorescent Proteins, or "TSFPs". Several green TSFPs are disclosed herein, as well as a unique methodology for generating such variants of other proteins, including fluorescent proteins. In particular embodiments disclosed herein, a class of TSFPs termed eCGPs are provided. The eCGPs of the invention were derived from an artificial fluorescent protein previously generated using a consensus engineering approach (consensus green fluorescent protein, or CGP44). The invention also provides polynucleotides encoding eCGPs, as well as vectors comprising such poynucleotides and cells transformed or containing such vectors. Various assay methods which utilize the TSFPs of the invention are also encompassed by the invention.

The TSFPs of the invention may be employed for all applications, methods and uses to which GFP and other fluorescent proteins are or may be applied, including their use as markers, as protein tags, in solubility screening, in the generation of split-CGP systems and assays, in protein trafficking and localization assays, in applications involving FRET, and the like. For example, TSFPs may be coupled to antibodies, polynucleotides or other receptors for use in detection assays such as immunoassays or hybridization assays, or to track the movement of proteins in cells. TSFPs may also be useful in systems to detect induction of transcription. For example, a nucleotide sequence encoding a TSFP can be fused to a promoter or other expression control sequence of interest, which can be contained in an expression vector, the construct can be transfected into a cell, and induction of the promoter (or other regulatory element) can be measured by detecting the presence or amount of fluorescence, thereby allowing a means to observe the responsiveness of a signaling pathway from receptor to promoter.

Because the TSFPs of the invention demonstrate remarkable stability in a number of challenging environments, they may find use in processes, assays and other applications in which a high degree of stability is required in order for the fluorescent phenotype to survive. For example, eCGPs show a remarkable degree of thermotolerance. Some of the eCGPs, such as eCGP23 and eCGP123, are able to retain fluorescence after being exposed to very high temperatures. For example, both eCGP23 and eCGP123 are able to recover almost completely after heating at 99°C, a temperature that irreversibly destroys folding in all other fluorescent proteins tested. Similarly, both of these eCGPs are able to retain some degree of fluorescence even at the high temperature of 990C. Additionally, both of these eCGPs retain approximately 85% of their ambient temperature fluorescence levels for at least 14 hours at 800C. Thus, these two eCGPs demonstrate remarkable and hitherto unreported levels of thermotolerance for fluorescent proteins, and should find use in various applications for which thermostability as well as increased stability generally are required. Detailed characterization of eCGPs is provided in the Examples which follow.

The fluorescence loss that occurs when fluorescent proteins are heated is caused by a combination of disruption of the local fluorophore environment caused by thermal vibrations and unfolding. Unfolding, in turn, can be either reversible or irreversible. In general, the loss of fluorescence caused by thermal vibrations is almost instantaneously reversible, that caused by reversible unfolding depends upon the kinetics of refolding, while irreversible unfolding does not recover. A number of lines of evidence indicate that most of the loss of fluorescence with heating eCGP123 to 990C is due to disruption of the local fluorophore environment, rather than unfolding of the protein. First, a thermal melt does not show the inflection point characteristic of the onset of cooperative unfolding, shown by the other proteins (FIG. 5a and 5b); secondly, at 990C, some residual fluorescence is clearly present for eCGP123 and eCGP23, while it is completely lost for the other proteins (FIG. 5b and 5c); thirdly, upon cooling after the thermal melt, over 60% of the fluorescence returns immediately (FIG. 5a and TABLE II); and finally, when the protein is repeatedly cycled between 99 and 300C fluorescence recovery is essentially immediate, and complete, with each cycle (FIG. 5c), while refolding would be expected to take longer. However, although it appears that most of the protein remains folded after short periods at 99°C, it is clear that prolonged incubations at high temperatures below 99°C can cause significant loss of fluorescence. After 14 hours at 8O0C, only 15% of the fluorescence normalized after stabilization at 80°C is lost (FIG. 5e), whereas at 85°C, only 15% of the fluorescence remains (FIG. 5f). When the thermal stability of the different evolved eCGP proteins is compared, the increased stability with increased evolution is striking, with the order of stability being eCGP123 > eCGP23 > eCGP13 > eCGP2 > eCGP1 > CGP: evolution around each additional loop, results in increased stability.

However, the individual loops are not equal in their stabilizing effects, with evolution around loop 2, appearing to provide the greatest individual stabilizing effect (compare eCGP1 to eCGP2 and eCGP13 to eCGP23). In fact, eCGP23 and eCGP123 are extremely similar to one another in their stability.

The eCGPs were also characterized by chemical denaturation using different concentrations of guanidine hydrochloride (FIG. 6a). See also, Example 1 , infra.

The invention further provides a method for generating stability-enhanced proteins. The method by which the eCGPs of the invention were generated is explained in detail in the Examples which follow. This method may be applied to other fluorescent proteins, and indeed, to virtually any protein, in order to generate increased-stability variants. Briefly, in the method of the invention, a recursive directed evolution strategy is employed, in which single destabilizing inserts are grafted into exposed loops of the protein in such a way that upon each insertion, folding and function are significantly affected but not destroyed (FIG. 1A). Upon overcoming the effect of a single insert by the initial round of evolution, the procedure is repeated with additional destabilizing inserts in an iterative fashion. The method enables one to overcome a final destabilizing force that would completely destroy both folding and function if applied in a single step.

The application of this method to a fluorescent protein is facilitated by the ease with which screening for correct folding can be carried out. However, this method is likely to be generally applicable to any protein, providing three criteria are fulfilled: 1 ) Surface exposed insert sites are correctly identified; 2) An appropriate destabilizing insert is used; and 3) A method to select correctly folded clones is available. In the example used herein, the structure of mAG, which was used to derive CGP, allowed the modeling and identification of the surface exposed loops. Although this is the ideal situation, when a structure or model are not available, it is possible that the application of secondary75' 76 or tertiary structural prediction methods77' 78 may provide sufficient information to identify suitable surface turns, since it is extremely unlikely that inserts placed within the protein core could be overcome by any degree of evolution.

The destabilizing insert used to generate the exemplified TSFPs disclosed herein here was based on an antibody heavy chain complementarity determining region 3 (HCDR3). This insert was chosen as the N and C termini of HCDR3s are close to one another within the context of an anti- parallel beta strand67, thereby presumptively providing destabilization without completely inhibiting folding. It is likely that alternative inserts could also provide appropriate degrees of destabilization, and it is possible that a panel of destabilizing inserts could be developed. In fact, such inserts could even comprise whole proteins in which the N and C termini were close to one another.

In the practice of the method of the invention, it is important to identify or develop a method to select or screen for correctly folded clones. When applied to fluorescent proteins, it is relatively straightforward to examine bacterial clones for fluorescence. A similar approach could be used for enzymes which can be expressed in bacteria, and for which colorimetric or fluorescent reagents are available. However, for the majority of proteins for which there is no obvious directly screenable phenotype, a separate screen for correct folding is required. This is not unlike the use of phage79"81 or yeast display82"85 to identify amino acids comprising specific binding sites: it is not sufficient to identify clones no longer binding to the binding partner, since loss of binding may be due to lack of folding. In addition to the negative selection for loss of binding, a positive selection for correct folding must also be included in the selection strategy. In the case of yeast, it is relatively straightforward, as only correctly folded proteins reach the cell surface and poorly folding proteins are retained in the endoplasmic reticulum. As a result it is sufficient to detect surface display using monoclonal83, polyclonal82 or anti- tag84 antibodies. In the case of phage display, recognition of conformational epitopes.

The invention also provides various methods which utilize TSFPs and TSFP coding sequences, such methods being currently employed with various other fluorescent proteins and variants thereof. For example, the invention provides a method for identifying the presence of a molecule in a sample. Such a method can be performed, for example, by linking a fluorescent protein variant of the invention to the molecule, and detecting fluorescence due to the fluorescent protein variant in a sample suspected of containing the molecule. The molecule to be detected can be a polypeptide, a polynucleotide, or any other molecule, including, for example, an antibody, an enzyme, or a receptor, and the like. The sample to be examined can be any sample, including a biological sample, an environmental sample, or any other sample for which it is desired to determine whether a particular molecule is present therein.

TSFPs may be linked to the molecule directly or indirectly, using any linkage that is stable under the conditions to which the protein-molecule complex is to be exposed. Thus, a TSFP and the subject molecule can be linked via a chemical reaction between reactive groups present on the protein and molecule, or the linkage can be mediated by linker moiety, which contains reactive groups specific for the fluorescent protein and the molecule. It will be appreciated that the appropriate conditions for linking a TSFP and the molecule are selected depending, for example, on the chemical nature of the molecule and the type of linkage desired. Where the molecule of interest is a polypeptide, a convenient means for linking a TSFP and the molecule is by expressing them as a fusion protein from a recombinant nucleic acid molecule, which comprises a polynucleotide encoding, for example, an eCGP operatively linked to a polynucleotide encoding the polypeptide molecule.

TSFPs may also be used in methods to identify agents and/or conditions that regulate the activity of an expression control sequence. Such methods may be performed, for example, by exposing a recombinant nucleic acid molecule, which includes a polynucleotide encoding a TSFP operatively linked to an expression control sequence, to an agent or condition suspected of being able to regulate expression of a polynucleotide from the expression control sequence, and detecting fluorescence of the TSFP due to such exposure. Such methods may be useful for identifying chemical or biological agents, including cellular proteins, that can regulate expression from the expression control sequence, including cellular factors involved in the tissue specific expression from the regulatory element. As such, the expression control sequence can be a transcription regulatory element such as a promoter, enhancer, silencer, intron splicing recognition site, polyadenylation site, or the like; or a translation regulatory element such as a ribosome binding site.

The invention also provides conservatively modified variants, as will be understood the those skilled in the art. Conservative substitutions may be tested using assays described herein or otherwise well known in the art. Other eCGP variant proteins can be identified, for example, using methods described in WO0123602 and other methods to select for increased folding. For example, to obtain an eCGP variant with increased folding ability, a "bait" or "guest" peptide that decreases the folding yield of the eCGP is linked to the eCGP. The guest peptide can be any peptide that, when inserted, decreases the folding yield of the eCGP, which may be measured by fluorescence, for example. A library of mutated fluorescent proteins is created. The bait peptide is inserted into the eCGP and the degree of fluorescence of the protein is assayed. Those clones exhibit increased fluorescence relative to a fusion protein comprising the bait peptide and parent eCGP are selected (the fluorescent intensity reflects the amount of properly folded fluorescent protein). The guest peptide may be linked to the eCGP at an end, or may be inserted at an internal site.

Various techniques for introducing mutations are well known in the art. These include, but are not limited to, such techniques as error-prone PCR, chemical mutagenesis, and cassette mutagenesis. Alternatively, mutator strains of host cells may be employed to add mutational frequency (Greener and Callahan, 1995, Strategies in MoI. Biol. 7: 32). For example, error-prone PCR (see, e.g., Ausubel, supra) uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Other mutagenesis methods include, without limitation, recombination, oligonucleotide-directed mutagenesis, phosphothioate-modified DNA mutagenesis, mutagenesis using uracil-containing templates, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair- deficient host strains, and deletion mutagenesis. Kits for mutagenesis are commercially available (e.g., Bio-Rad, Amersham International). More recent approaches include codon-based mutagenesis, in which entire codons are replaced, thereby increasing the diversity of mutants generated, as exemplified by the RID method described in Murakami et al., 2002, Nature Biotechnology, 20: 76-81.

The TSFP polypeptides may be prepared using methods well known in the art, including by peptide synthesis and recombinant production means. For example, an eCGP may be synthesized according to standard solid-phase methodologies, utilizing the amino acid sequences provided herein, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer

(Applied Biosystems, Foster City, Calif.), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art.

Also provided are vectors containing the TSFP polynucleotides of the invention, as well as host cells transformed or transfected with, or otherwise made to contain, such vectors. Also provided is a recombinant nucleic acid molecule, which includes at least one polynucleotide encoding a TSFP operatively linked to one or more other polynucleotides. The one or more other polynucleotides can be, for example, a transcription regulatory element such as a promoter or polyadenylation signal sequence, or a translation regulatory element such as a ribosome binding site. Such a recombinant nucleic acid molecule can be contained in a vector, which can be an expression vector, and the nucleic acid molecule or the vector can be contained in a host cell. A vector of the invention will generally contain various elements required for replication in a prokaryotic or eukaryotic host system, or both, as required. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a number of commercial sources or constructed using methods well known in the art.

The disclosed eCGPs, eCGP variants, or fusions of an eCGP and another polypeptide, may conveniently expressed in a suitable host cell, such as an E. coli cell, using an eCGP-encoding polynucleotide, such as the DNA coding sequences for eCGPs provided in the TABLE OF SEQUENCES, infra.

There are many expression systems for producing the proteins of the invention that are well know to those of ordinary skill in the art. (See, e.g., Gene Expression Systems, Fernandes and Hoeffler, Eds. Academic Press, 1999; Russell & Sambrook, supra). Commonly used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter, the tac promoter and the lambda-derived PL promoter and N-gene ribosome binding site. The particular promoter system is not critical to the invention, any available promoter that functions in prokaryotes can be used. Standard bacterial expression vectors include plasmids such as pET, pTET, pBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, λ-phage derived vectors, p15A-based vectors and fusion expression systems such as GST. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltose binding protein, VSV-G tag, anti-DYKDDDDK tag, or any such tag, a large number of which are well known to those of skill in the art.

For expression of fusion polypeptides in prokaryotic cells other than E. coli, regulatory sequences for transcription and translation that function in the particular prokaryotic species is required. Such promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used. For example, the hybrid trp-lac promoter functions in Bacillus in addition to E. coli. These and other suitable bacterial promoters are well known in the art and are described, e.g., in Russell & Sambrook and Ausubel et al. Bacterial expression systems for expressing the proteins of the invention are well known and commercially available.

Similarly, the for expression of fusion polypeptides in eukaryotic cells, transcription and translation sequences that function in the particular eukaryotic species are required. For example, eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In yeast, vectors include Yeast Integrating plasmids (e.g., Ylp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-

Barr virus. Other exemplary eukaryotic vectors include those employing the

CMV promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

Either constitutive or regulated promoters can be used in the present invention. Regulated promoters can be advantageous because the host cells can be grown to high densities before expression of the polypeptide is induced. High level expression of heterologous proteins slows cell growth in some situations. An inducible promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals.

Inducible promoters for other organisms are also well known to those of skill in the art. These include, for example, the metallothionein promoter, the heat shock promoter, as well as many others. Translational coupling may be used to enhance expression as is well known.

The strategy uses a short upstream open reading frame derived from a highly expressed gene native to the translational system, which is placed downstream of the promoter, and a ribosome binding site followed after a few amino acid codons by a termination codon. Just prior to the termination codon is a second ribosome binding site, and following the termination codon is a start codon for the initiation of translation. The system dissolves secondary structure in the RNA, allowing for the efficient initiation of translation.

The construction of polynucleotide constructs generally requires the use of vectors able to replicate in host bacterial cells, or able to integrate into the genome of host bacterial cells. Such vectors are commonly used in the art. A great number of systems and kits are commercially available for the purification of plasmids from bacteria (for example, EasyPrepJ, FlexiPrepJ, from Pharmacia Biotech; StrataCleanJ, from Stratagene; and, QIAexpress Expression System, Qiagen). The isolated and purified plasmids can then be further manipulated to produce other plasmids, and used to transform cells.

The TSFP polypeptides can be expressed intracellular^, or can be secreted from the cell. Intracellular expression often results in high yields. If necessary, the amount of soluble, active fusion polypeptide may be increased by performing refolding procedures (see, e.g., Sambrook et al., supra.; Marston et al., Bio/Technology (1984) 2: 800; Schoner et al., Bio/Technology (1985) 3: 151). Fusion polypeptides of the invention can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The host cells can be mammalian cells, insect cells, or microorganisms, such as, for example, yeast cells, bacterial cells, or fungal cells.

Once expressed, the recombinant polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)).

To facilitate purification of the TSFP polypeptides of the invention, the nucleic acids that encode the fusion polypeptides can also include a coding sequence for an epitope or "tag" for which an affinity binding reagent is available. Examples of suitable epitopes include the myc and V-5 reporter genes; expression vectors useful for recombinant production of fusion polypeptides having these epitopes are commercially available (e.g., Invitrogen (Carlsbad CA) vectors pcDNA3.1/Myc-His and pcDNA3.1/V5-His are suitable for expression in mammalian cells).

Additional expression vectors suitable for attaching a tag to the fusion proteins of the invention, and corresponding detection systems are known to those of skill in the art, and several are commercially available (e.g., FLAG" (Kodak, Rochester NY). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity ligands. Typically, six adjacent histidines are used, although one can use more or less than six. Suitable metal chelate affinity ligands that can serve as the binding moiety for a polyhistidine tag include nitrilo-th-acetic acid (NTA) (Hochuli, E. (1990) "Purification of recombinant proteins with metal chelating adsorbents" In Genetic Engineering: Principles and Methods, J. K. Setlow, Ed., Plenum Press, NY; commercially available from Qiagen (Santa Clarita, CA)).

Various aspects of the invention are further described and illustrated by way of the several examples which follow, none of which are intended to limit the scope of the invention. EXAMPLES



CDR3 insertions into CGP

The 60-bp CDR3 sequences were inserted into CGP by PCR assembly. The PCR primers generally contained a 20-bp long CGP specific sequence and a 40-bp 5' tail encoding one part of the CDR3. The two CDR3 containing primers had a 20-bp homologous overlapping sequence to facilitate PCR assembly. The following general procedure was used, unless otherwise described. The reaction was performed in 50 μl containing 1x Thermopol buffer (NEB), 250 μM dNTP, 0.5 μM of each primers and 1.25 U Taq polymerase (NEB) with cycling conditions as follows: 1 min initial denature at 94 0C, then 30 cycles of 94 0C, 15 sec, 55 0C, 15 sec, 30 sec, 72 0C, then a final elongation of 5 min at 72 0C. TABLE IV contains the primer sequences. The nucleotide sequence encoding the 20 amino acid long CDR3 sequence was different for each insert, using different codons, in order to prevent homologous recombination in the bacteria. Inserting single CDR3s into CGP was achieved by performing two PCR reactions with 1) CGP-5' and CDR- loop-x-R-CGP and 2) CDR-loop-x-F-CGP and CGP-3' primers. The x denotes the loop number. The resulting bands were gel purified and assembled in an assembly reaction with CGP-5'and CGP-3' primers added after 25 cycles.

Multiple CDR3s were inserted similarly. The double inserted libraries were assembled from 3 fragments; the triple inserted libraries used 4 fragments. For example the double library containing CDR3 insert in loop 1 and loop 3 were assembled from fragments amplified using these primers: 1) CGP-51 and CDR-loop-1-R-CGP; 2) CDR-loop1-F-CGP and CDR-loop-3-R-CGP; 3) CDR- loop-3-F-CGP and CGP-3' (see TABLE IV). Other libraries were created similarly using the appropriate primers.

DNA shuffling DNA shuffling was performed according to Zhao, 199791. Briefly, 10 μg of template DNA, CGP containing 1 , 2 or 3 CDR3 inserts, were digested with 1 U of Dnasel (NEB) for 10 minutes at 15 0C in 50 mM Tris-Ac pH 7.5, 2 mM C0CI2. The reaction was terminated by heating for 3 minutes at 90 0C, and DNA fragments purified by spin-column chromatography on Sephadex-25 (GE Healthcare) columns. The digested template was assembled in a primerless PCR reaction with 1.25 U Pfu Exo" DNA polymerase (Stratagene) using 15 μl of the digested template in a buffer containing 1x Pfu reaction buffer, 0.4 mM dNTP in a 25 μl reaction volume. The cycling conditions were 97 0C, 3 min initial denature, then 35 cycles of 96 0C 25 sec, 56 0C, 25 sec, 72 0C 1 min, with a final extension for 5 min at 72 0C. 3μl of the amplification reaction was amplified by 2.5 U Taq polymerase (NEB) in a 100 μl reaction containing 1x Thermopol buffer (NEB), 250 μM dNTP, 0.5 μM of CGP-5' and CGP-3' primers with the following cycling: 1 minute initial denature at 94 0C, then 30 cycles of 94 0C, 15 sec; 60 0C, 15 sec; 72 0C 30 sec; with a final elongation for 5 minutes at 72 0C. The PCR product was phenol/chloroform extracted and purified by spin-columns containing Sephadex G-75 (GE Healthcare). The purified DNA was digested with BssHII (NEB) and Nhel (NEB) according to the manufacturer's recommendation and cloned into pETCK3 (Kiss et al., 200688).The ligation was electroporated into BL21 (DE3) Gold electrocompetent cells. The cells were plated on nitrocellulose filters on LB agar plates containing 50 μg/ml kanamycin and 3% glucose and grown overnight at 37 0C. The filters were transferred onto kanamycin LB plates containing 1 μg/ml IPTG and induced for 4 hours at 30 0C. Colonies that were greenest after induction were picked and sequenced. The selected clones for the next round of shuffling were pooled and the CDR3 sequences were recreated by PCR assembly using CDR3 specific primers that lacked any CGP specific sequences. Protein Expression and Purification

Plasmids encoding the fluorescent proteins cloned into pETCK3 were transformed into E.coli BL21 DE3 cells (Stratagene). Single colony transformants were cultured overnight at 37° in Luria Broth with 50μg/ml kanamycin. The overnight cultures were suspended in fresh Terrific Broth containing 50μg/ml Kanamycin and transferred to the Kalypsis Airlift Fermentation System, based on the system described by Lesley et al89. Cultures were grown at 37° for 3 hours (optical density of 1.5 - 2.5 (600nm)) on 100% air. The temperature was reduced to 30° and IPTG added to a final concentration of 1 mM. After 4 hour of growth, 50% air and 50% oxygen, cells were harvested by centrifugation and the resulting pellets were stored overnight at -20°. The bacteria pellets were removed from storage, thawed, and suspended in lysis buffer (50OmM NaCI, 5mM Imidazole). Cells were lysed by sonication in the Kalypsis pre-chilled rotor, using 4 cycles of one minute sonication (duty cycle 100, amplitude 75) followed by one minute rest, then centrifuged at 7000 x g for 30 min. The Kalypsis Robot transferred the supernatant to the nickel columns (Nickel Chloride bound to GE Chelating Sepharose Fast Flow Resin) which were washed with (50OmM NaCI, 5mM Imidazole). The bound proteins were eluted with (50OmM NaCI, 50OmM Imidazole).

The fluorescence of the purified proteins was measured (SPECTRAFIuor Plus, 492 nm, optimal gain 44) in arbitrary fluorescence units measured at 535 nm. An SDS-PAGE gel was loaded with samples based on equal fluorescence and proteins were quantified against protein standards using the Syngene GeneTool Software.

Thermostability measurements Proteins of equal fluorescence were diluted into 50 μl of TNG buffer (100 mM Tris-Ac pH 7.5, 100 mM NaCI, 10 % glycerol) and placed into 0.2 ml thin wall PCR tubes. Thermal cyclings were performed in a Rotor-Gene 6000 real time PCR machine (Corbett Life Science). Fluorescence and gain were adjusted so that the fluorescence of the starting samples was between 90-100. The melting profile was resolved between 30 0C and 99 0C. Temperature was raised by 0.5 0C increments. The samples were incubated at each temperature for 60 sec.

Single molecule spectroscopy

Fluorescence Correlation Spectroscopy was performed in the same setup described previously44. Quantum yield was determined relative to Fluorescein from the ratio of integrated fluorescence signal to the absorbance at 488 nm.

Chemical denaturation

Equilibrium fluorescence values were measured by diluting guanidine hydrochloride denatured eCGP variants into TNG containing 5 mM DTT to various final guanidine concentrations between 1 and 8 M in increments of 0.15 M guanidine, and allowing refolding to proceed at 15°C. Fluorescence values were measured using a FL600 Microplate Fluorescence Reader (488- nm excitation, 530-nm emission, 10-nm band pass) and scaled by dividing by the fluorescence levels of corresponding nondenatured samples diluted in parallel as a reference. Midpoint recovery concentrations of guanidine Cm (recovery of 50% of the initial fluorescence) were determined from sigmoidal fits using SOLVER in EXCEL, to the scaled fluorescence value F using the equation Fj 1/4 a + b/(1 + (Cj/Cm)h), where a, b, Cm and h are adjustable parameters, and Cj is the molarity of the guanidine in the refolding experiment j. The data were used to calculate the dependence of the standard free energy of denaturation, DG1 1/4 -RT In K, on guanidine concentration, where R is the gas constant, T is the absolute temperature and K is the equilibrium constant, which can be calculated from the experimental data by using the standard equation K 1/4 [(y)N - (y)]/[(y) - (y)D], where (y) is the observed value of the parameter used to follow unfolding, and (y)N and (y)D are the (y) values for the native state and the denatured state, respectively, under the same conditions under which (y) was measured. RESULTS Evolutionary strategy

A recursive evolutionary strategy was employed, in which single inserts were grafted into exposed loops in such a way that upon each insertion, folding and function were significantly affected but not destroyed (FIG. 1A). This provides a baseline which may be improved by evolution. Upon overcoming the effect of a single insert, the procedure is then repeated with a second, and finally a third insert. In this way it is possible to overcome a final destabilizing force that would completely destroy both folding and function if applied in a single step.

This method was applied to CGP by modeling the structure of this protein on that of Dronpa, the closest fluorescent protein for which a structure has been determined (Wilmann et al., 200690), and targeting three identified beta turns for insertion. These were termed loop 1 (V18/N19), loop 2 (E96/D97) and loop 3 (E164/G165). The destabilizing insert used was based on a human heavy chain third antibody omplementarity determining region (HCDR3) sequence. Although HCDR3s are highly diverse loops, they are embedded into a relatively conserved beta sheet structure66, as a result of which the amino acids at either end (cysteine 104 and tryptophan 119; IMGT numbering67 are always joined by two hydrogen bonds. As the usual distance between these two amino acids is similar to that between amino acids just before the turns described above, it was presumed that the insertion of such a sequence into a CGP loop would probably be disruptive to folding, but would not destroy it completely. In order to avoid the presence of an unpaired cysteine (the HCDR3 N terminal cysteine normally disulfide bonds with another cysteine in framework one), this codon was mutated to a serine, which is able to form the same hydrogen bonds. The final sequence used (SARSFYLQSDLAAGDFDSWG) based on a randomly picked HCDR3 with a few internal changes to facilitate cloning, was inserted at V18/N19 and E96/D97 in two independent PCR assemblies. As expected, this resulted in a significant reduction in the fluorescence of induced bacterial colonies as shown in FIG. 1 B. After three rounds of error prone PCR and DNA shuffling on these two modified genes, the fluorescence of induced bacterial colonies increased significantly, some reaching the levels of the original CGP protein (FIG. 1 B). During the mutation and selection process, the gene was always reassembled using the HCDR3 as an anchor, in order to force mutations into the CGP and not the inserted HCDR3. After three rounds, PCR assembly was again used to insert the same HCDR3 amino acid sequence at position E164/G165 of the genes from 23 fluorescent evolved clones containing an insert at V18/N19 and 22 clones containing an insert at E96/D97. The DNA sequence encoding the HCDR3 insert was altered to avoid recombination with the first insert. A significant reduction in fluorescence was again observed, which could be restored after three further rounds of evolution, carried out as before by PCR assembly of now three fragments using the two HCDR3 inserts as anchors.

The process was repeated a final time, pooling fluorescent colonies and inserting the HCDR3 into three sites (V18/N19, E96/D97 and E164/G165). With the proteins containing three inserts, four rounds of evolution were required before fluorescence was significantly restored. After each round of evolution approximately 100 clones were sequenced, allowing analysis of the accumulated mutations (FIG. 2). It should be pointed out that although the use of assembly PCR to insert each additional loop into CGP allowed mutations accumulated in previous rounds to persist into the following evolutionary rounds, this was not true of those mutations close to the insert site, which were "overridden" by the primers used for insertion in the first round. In subsequent rounds, the HCDR3 insertion sequences themselves were used for assembly, allowing reappearance of mutations close to insertions. In general 4 classes of mutations were observed: 1) those (e.g. D7E, M40L, T59P, V60A) appearing immediately and retained throughout; 2) those which first appear with a single insert, are specific for that insert (e.g. Q98H for 18/19 inserts, and K22E for 96/97 inserts), are retained in the presence of two inserts, but are then lost when three inserts are present; 3) those appearing in the presence of two inserts, and persisting in the presence of three inserts (e.g. E164K, K190E, K208R); and 4) those (e.g. A17S, K30I, F34Y, A53S) which are only found when three inserts are present. Gene synthesis

Genes corresponding to the proteins without inserts were synthesized (Blue Heron Biotechnology) for each of the five evolutionary paths (FIG. 1A). Synthesized genes contained those mutations that led to amino acid changes in at least 20% of sequences, and silent mutations found in greater than 90% of sequences. In addition, one silent mutation frequently found adjacent to a non-silent mutation was also included. In order to concentrate on mutations responsible for global increases in stability, rather than mutations responding to specific changes in secondary structure adjacent to the insert site, those mutations found within two amino acids of an insertion point were not included, even though there are examples of mutations in loops (e.g. Y39N in sfGFP7) which are globally stabilizing.

The aligned amino acid sequences of the final genes synthesized, compared to CGP, are shown in FIG. 3. As can be seen, some mutations (eight of eighteen) recapitulate amino acids found in fluorescent proteins used to create the CGP consensus sequence. The remaining ten mutations are equally split between those found in mAG (and modified for CGP) and those not previously found in any other fluorescent protein, and unique to these evolved proteins. Of the mutations which revert back to mAG, three (D7E, M40L, A69T) are found in most of the evolved proteins, while the remaining two (K32N and F34Y) are each found in only one or two of the proteins. The reversion of such presumably destabilizing mutations in consensus sequences is similar to those found in other examples37"*0, and underlie the importance of examining the roles of individual amino acids for their contributions to stability.

Properties of eCGPs The five fluorescent protein genes were cloned into pETCK388 and expressed in BL21. All were able to direct the synthesis of fluorescent proteins at levels comparable to, or exceeding, CGP and mAG (FIG. 4a). The excitation / emission properties (FIG. 4b and TABLE I) of the proteins were similar to either CGP (eCGP1 and eCGP2) or mAG (eCGP13, eCGP23 and eCGP123), with the CGP series being slightly red shifted compared to the mAG series. The quantum yields of the proteins ranged from 0.54 (eCGP1) to 0.75 (eCGP13), not too dissimilar to that of mAG (0.83). All proteins were monomeric as determined by gel filtration (not shown) or fluorescence correlation spectroscopy (TABLE I).

In a first test of protein thermostability, the proteins were slowly melted at 0.5 "C/rnin, using a real time PCR machine (Rotor-Gene 6000, Corbett Life Sciences, FIG. 5a) which monitored fluorescence changes with temperature in real time. The temperature was gradually increased to 99°C, and then returned to 3O0C, to monitor recovery. After approximately 380C, all proteins showed a reduction in fluorescent with increasing temperature as shown in FIG. 5a. This fluorescence loss is characteristic of fluorescent proteins, and thought to be due to two components: changes in the immediate fluorophore environment caused by increased thermal vibrations, and unfolding of the proteins. Fluorescence loss due to the former are immediately reversible and do not represent unfolding68' 69, while fluorescence loss due to the latter require refolding for fluorescence to return. As temperature increases, the proportion of fluorescence loss due to these two components will vary, depending upon the stability of the protein and the temperature. In general, little of the fluorescence loss is caused by unfolding until the temperature at which cooperative unfolding starts is reached. This is recognized as an inflection point in the melting curve, and represents the point at which unfolding suddenly accelerates. This is similar to changes in CD spectra observed with increasing temperature70.

All proteins, with the exception of eCGP23 and eCGP123, showed cooperative unfolding as the temperature was increased, with inflection points between 73 and 87°C, cooperative transition midpoints two to three degrees later, and characteristic steeper denaturation curves69. eCGP23 and eCGP123 were characterized by the absence of a clear cooperative transition, and even at 99°C, some fluorescence remained (FIG. 5b). Recovery upon cooling to 30°C resulted in essentially complete (96%) recovery of eCGP123, and 85% recovery of eCGP23 (TABLE II). The remaining proteins recovered to varying degrees, depending upon the degree of evolution. For all the evolved proteins, 54-61 % of the fluorescence recovery occurred instantaneously, while for mAG and CGP, the instant recovery was lower (35% and 44% respectively).

The same order of stability was observed when the proteins were treated with multiple heat cool cycles (equivalent to 60 "PCR cycles" with 1 minute denaturation at 99°C and 2 minutes recovery at 300C - FIG. 5c for CGP and eCGP123). eCGP123 and eCGP23 continued to show low levels of fluorescence at 99°C, while the other proteins rapidly lost fluorescence at this temperature. After 60 heat/cool cycles, and at each return to 3O0C, the fluorescence of the two stable proteins returned to their pretreatment levels, while the remaining proteins showed a dramatic drop after the first heat cycle, with fluorescence further decreasing to zero with additional cycles, and little recovery upon return to 300C.

One last test of thermal stability was the ability of the proteins to resist high temperature for prolonged periods. The proteins were all heated to 800C or 85°C. This resulted in the initial rapid loss of over 80% fluorescence due to thermal vibration, which stabilized after about six to seven minutes. The fluorescence of the different proteins was normalized at this time (arrow FIG. 5d), and further fluorescence loss monitored for 14 hours. eCGP123 and eCGP23 lost approximately 15% fluorescence after 14 hours at 8O0C, while all the other proteins, with the exception of eCGP13 which was intermediate, had lost all fluorescence by 2-3 hours (FIG. 5e). At 850C the fluorescence loss of the less stable proteins (CGP, eCGP1 and mAG) was complete by five minutes. eCGP1 and eCGP13 showed complete loss of fluorescence by three hours, while after 14 hours eCGP23 and eCGP123 still retained approximately 10-15% of the normalized fluorescence at 85°C (FIG. 5F).

Thermal denaturation was monitored using measures independent of intrinsic fluorescence. However, the Thermofluor assay71' 72 was unsuccessful due to degradation of the Sypro Orange at temperatures above 800C, and it also proved impossible to carry out circular dichroism at the high temperatures required. eCGP stability was also studied by denaturation in guanidine hydrochloride (FIG. 6A and TABLE III) with unfolding monitored by fluorescence. At equilibrium, which required over two weeks, eCGP123 and eCGP23 were again the most stable proteins, with melting (kd) occurring at 6.45 M guanidine for eCGP123 and 6.19 M for eCGP23. However, the order of stability for the remaining proteins was slightly different to that observed with thermal denaturation, with eCGP2 being significantly more stable than eCGP13, and CGP being more stable than eCGPL By extrapolating a natural log fit of the sigmoidal denaturation curve to infinite dilution (FIG. 6B), the ΔG was determined, which again showed eCGP123 to be by far the most stable protein at 12.4 kcal/mol.

CGP, mAG, and eCGP123, representing the starting, evolved, and closest natural proteins, were also analyzed for folding kinetics. Proteins were denatured in Gdn HCI, and fluorescence recovery monitored upon dilution into fresh buffer. Although CGP is much less stable than mAG, it displayed an approximately 3.5-fold faster initial rate for fluorescence recovery relative to the more stable mAG (FIG. 6C, inset). This faster folding behavior is consistent with the observation that CGP also unfolds much faster than mAG in 8 M Gdn HCI as noted above. Such behavior is typical of simple two-state folders, for which increased forward folding rate is mirrored by a corresponding increased unfolding rate.

eCGP123 folds 4-fold faster than CGP. The increased stability of eCGP123 relative to CGP likely results from the very slow unfolding of eCGP123 consistent with the slow approach to equilibrium during the equilibrium Gdn HCI unfolding experiments and the thermal stability. TABLES

Figure imgf000039_0001


Figure imgf000039_0002


Figure imgf000039_0003

Figure imgf000040_0001


eCGP DNA Sequences : eCGPl [ SEQ ID NO : 1 ]
















eCGP Amino Acid Sequences : eCGPl [SEQ ID NO: 6]







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I . A polypeptide comprising the amino acid sequence of SEQ ID NO: 9.
2. A polypeptide having the amino acid sequence of SEQ ID NO: 9.
3. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide according to claim 1.
4. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide according to claim 2.
5. The nucleic acid molecule of claim 3 or 4, wherein the amino acid sequence of SEQ ID NO: 9 is encoded by the polynucleotide of SEQ ID NO: 4.
6. A polypeptide comprising the amino acid sequence of SEQ ID NO: 10.
7. A polypeptide having the amino acid sequence of SEQ ID NO: 10.
8. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide according to claim 6.
9. A nucleic acid molecule comprising a polynucleotide encoding the polypeptide according to claim 7.
10. The nucleic acid molecule of claim 8 or 9, wherein the amino acid sequence of SEQ ID NO: 10 is encoded by the polynucleotide of SEQ ID NO: 5.
I I . A vector comprising the nucleic acid molecule of any one of claims 3, 4, 5, 8, 9 and 10.
2. A cell comprising a vector according to claim 11.
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