# Patent

## Title
Identification and characterization of reflectin proteins from squid reflective tissues

## Abstract

A family of reflectin proteins is identified herein that is deposited in flat, structural platelets in reflective tissues of the squid Euprymna scolopes. These proteins are encoded by at least six genes in three subfamilies and have no reported homologues outside of squids. Reflectins possess 5 repeating domains, that are remarkably conserved among members of the family. The proteins have a highly unusual composition with four relatively rare residues (tyrosine, methionine, arginine, and tryptophan) comprising  ̃57% of a reflectin, and several common residues (alanine, isoleucine, leucine, and lysine) occurring in none of the family members. These protein-based reflectors in squids provide a striking example of nanofabrication in animal systems.

## Background
PRIORITY
This application claims priority to U.S. Provisional Application 60/549,733, filed Mar. 2, 2004, herein incorporated by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
This invention was made in part with Government support under Grant No. IBN 0211673, and AI50661 awarded by the National Institutes of Health. The Government may have certain rights in this invention.


FIELD OF THE INVENTION
The invention relates generally to a new family of proteins that compose a subcellular structure that confers reflectivity to squid tissues. More specifically, the invention relates to squid reflectin proteins and active portions and repeat units thereof.
BACKGROUND OF THE INVENTION
The biological world is an arena of nanofabrication, one that can be tapped for information about constraints on the design and production of small-scale materials. Among the most intricate of natural nanoscale materials are those that modulate light, such as the lenses, irises, and reflectors of animals (Vukusic, et al. 2003 Nature 424, p. 852). Reflective tissues are prevalent across the animal kingdom, being particularly conspicuous in species that live in the visually homogeneous pelagic environments of the ocean. In these habitats, reflectors often function in camouflaging by modulating incident sunlight or bioluminescence (Johnsen, et al. Proc. Royal Soc. London. B 2001, 269, p. 243; Johnsen, et al. Limnol. Oceanogr. 2003, 48, p. 1277). Reflectivity in animal tissues is achieved by the deposition of flat, insoluble, structural platelets of high refractive index that alternate in layers with materials of low refractive index. This arrangement creates thin-film interference, that results in reflection of some or all of the incident light (Land, et al. Prog. Biophys. Molec. Biol. 1972, 24, p. 75). In aquatic animals, reflector platelets are most often composed of purine crystals, particularly guanine and hypoxanthine (Denton, et al. Proc. Roy. Soc. Lond. A. 1971, 178, 43). In contrast, cephalopod reflector platelets do not contain these purines and studies of their biochemical and biophysical characteristics have suggested that they are composed of protein (Cooper, et al. Cell Tissue Res. 1990, 259, p. 15). However, the composition of cephalopod reflector platelets has never been definitively characterized (Cloney, et al. Amer. Zool., 1983, 23, p. 581). Each of the cited references herein are incorporated by reference in its entirety.
SUMMARY OF THE INVENTION
One embodiment provides for an isolated reflectin polypeptide having a sequence with at least about 75% identity to SEQ ID NO:2, including 85% identity to SEQ ID NO:2. In some embodiments, the polypeptide has a predicted isoelectric point above 8.0.
Other embodiments provide for an isolated polynucleotide encoding a reflectin polypeptide, the polynucleotide having a sequence with at least about 65% identity to SEQ ID NO:1, including 77% identity and 85% identity.
Other embodiments provide for an isolated polypeptide having and least one and no more than four repeats of an amino acid sequence having the motif [α(X)4/5MD(X)5MD(X)3/4], wherein α is MD, FD, or null; X represents any amino acid; the subscripted numbers represent the number of amino acids at that position; and the slash represents “or.” In some embodiments the amino acid sequence is selected from the group consisting of: SEQ ID NOs: 15-30 and any combination thereof. In other embodiments the isolated polpeptide has an activity of a reflectin protein.
Other embodiments provide for an isolated polypeptide having six or more repeats of an amino acid sequence having the motif [α(X)4/5MD(X)5MD(X)3/4], wherein α is MD, FD, or null; X represents any amino acid; the subscripted numbers represent the number of amino acids at that position; and the slash represents “or.” In some embodiments the amino acid sequence is selected from the group consisting of: SEQ ID NOs:15-30, and any combination thereof.
Other embodiments provide for a biomimetic reflective material having a first component, the first component having at least one polypeptide selected from (a) a reflectin polypeptide; (b) a polypeptide having at least one and not more than four repeat units of a reflectin polypeptide; (c) a polypeptide having at least six repeat units of a reflectin polypeptide; (d) an active or functional homologue or recombinant form of any of (a) through (c); and (e) any combination of (a) through (d); the first component being in combination with at least a second component compatible with the first component, such that the combination forms a biomimetic reflective material. In some embodiments, the biomimetic reflective material includes a metal ion. In other embodiments, the material has at least a first and a second refractive state, wherein the material in the first refractive state has a refractive index that is different from a refractive index of the material in the second state.
Other embodiments provide for a method of producing a biomimetic reflective material, by providing a first component having at least one polypeptide (a)-(e) where (a) a reflectin polypeptide; (b) a polypeptide having at least one and not more than four repeat units of a reflectin polypeptide; (c) a polypeptide having at least six repeat units of a reflectin polypeptide; (d) an active or functional homologue or recombinant form of any of (a) through (c); and (e) any combination of (a) through (d); combining the first component with at least a second component to form a biomimetic reflective material. In some embodiments, the second component is a metal, an ion, a polymer, a fabric, a crystal, a fiber, a plastic or any other suitable material.
Further embodiments include a method of producing a biomimetic reflective material, by causing expression in a cell, of at least one polypeptide selected from: (a) a reflectin polypeptide; (b) a polypeptide having at least one and not more than four repeat units of a reflectin polypeptide; (c) a polypeptide comprising at least six repeat units of a reflectin polypeptide; (d) an active or functional homologue or recombinant form of any of (a) through (c); and (e) any combination of (a) through (d); using the cell or a fragment or extract thereof in producing a biomimetic reflective material. In some embodiments, the cell is a plant cell, a bacterial cell; a fungal cell, or an animal cell.
Further embodiments provide for a method of modifying a refractive index of a reflectin, by: providing a reflectin polypeptide in a composition compatible with a metal ion, wherein the composition has a first refractive index in absence of the metal ion; adding the metal ion to the composition, wherein the composition has a second refractive index in presence of the metal.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Reflective tissues in Euprymna scolopes. (A) The locations of reflective (dg, digestive gland; er, eye reflector; lor, light organ reflector; m, mantle) and non-reflective (el, eye lens; g, gill) tissues of E. scolopes are revealed by a ventral dissection of an adult animal; inset, an adult animal. (B) A light micrograph of a cross section (location at orange line in panel A) of the light organ. The central epithelium (e) is surrounded by the reflector (lor), that is in turn surrounded by ink sac diverticula (is). Lens tissue (lol) is located on the ventral surface of the light organ. (C) Transmission electron micrograph (TEM) of the boxed area in B. Stacks of electron-dense reflector platelets (p) abut connective tissue (ct) and the ink sac with its secreted ink granules (ig). (D) Higher magnification TEM of the light organ reflector (LOR) platelets. (E) Silver-stained SDS-PAGE gel of protein extracts from LORs. LOR tissue extracts were prepared as described in the Examples. lane 1, molecular mass markers (std) expressed in kDa; lane 2, total homogenate of LOR (total); lane 3, supernatant fraction of LOR extracted in PBS (soluble); lane 4, supernatant of pellet from PBS-extracted LOR re-extracted with 2% SDS (pellet); lane 5, purified reflectins (purified). 5 μg of protein loaded in lanes 2-4 samples; 3 μg loaded in lane 5. (E′) Higher magnification of reflectin bands, showing the presence of 3 protein species.
FIG. 2. Localization of reflectins in E. scolopes. (A) Immunocytochemistry at the light level. One-μm sections of whole juvenile squid mounted on gelatin-coated glass slides were incubated in either preimmune serum (preimmune) or reflectin antiserum (anti-reflectin). Fifteen-nm gold beads conjugated to goat anti-rabbit IgGs were used as secondary antibodies and sections were silver enhanced (Silver Enhancer Kit; Sigma-Aldrich) to allow detection of the gold beads by light microscopy. The sections were then either counterstained with 1% acid fuchsin (+cs) or left unstained (−cs) and viewed by differential interference microscopy. is, ink sac; lor, reflector; e, central epithelial tissue. (A′) Immunocytochemistry at the TEM level. Ultrathin sections of whole juvenile squid mounted on nickel grids were incubated in either preimmune serum or reflectin antiserum. Fifteen-nm gold beads conjugated to goat anti-rabbit IgGs were used as secondary antibodies. Inset, higher magnification showing the labeling of an individual platelet. p, platelets; ct, connective tissue; ig, ink granules. (B) Silver-stained SDS-PAGE (lanes 1-9) and immunoblot analyses (lanes 10-18) of 2% SDS-extracted proteins from pellets of aqueous-buffer extractions of reflective and non-reflective squid tissues; 2.5 μg total protein loaded per lane. std, molecular mass standards in kDa; arrowhead indicates the position on the gels where reflectins resolve. dig. gland, digestive gland; l.o. lens, light organ lens; l.o. reflector, light organ reflector.
FIG. 3. Reflectins are composed of repeating domains, as predicted by RADAR (Rapid Detection and Alignment of Repeats). (A) Upper panel, the entire amino acid sequence of reflectin 1a with repeat regions indicated in grey and amino acids excluded from the repeats in black (SEQ ID NO:2). Numbers and arrows indicate the number and direction of the repeats. Lower panel, the RADAR output. The fourth repeat was used as the template repeat. Score, “score of each repeat unit when scored against the whole repeat” (RADAR); Std Dev, the number of standard deviations above the mean for shuffled sequences scored against the same profile. (B) Schematic showing the positioning of the repeats (gray boxes) and the conserved subdomains (hatched boxes; SD1-SD5) from a representative reflectin. The subdomains are also outlined in the RADAR alignment in panel 3A (SEQ ID NOs:16-24). The subdomain amino acid alignments among all reflecting are shown under each subdomain. SD1=SEQ ID NO:16, SD2=SEQ ID NO:17, SD3=SEQ ID NO:18 and 19, SD4=SEQ ID NO:20 and 21, and SD5=SEQ ID NOs: 22, 23, and 24.
FIG. 4. Amino acid alignments, comparisons, and compositions of the derived amino acid sequences for E. scolopes reflecting and L. forbesi mrrp1 (Lf). (A) Alignment (Clustal V) of reflectin proteins 1a-3a with L. forbesi mrrp1. 1a is SEQ ID NO:2, 1b is SEQ ID NO:4, 2a is SEQ ID NO:6, 2b is SEQ ID NO:8, 2c is SEQ ID NO:10, 3a is SEQ ID NO:12, and Lf is SEQ ID NO:14. The black bars are located above the tryptic peptides that were sequenced (SEQ ID NOs: 31-33); *, residues are identical in all sequences in the alignment; colon (:), are conserved substitutions present in the alignment; period (.), are semi-conserved substitutions present in the alignment. (B) Pairwise comparison of the reflectins and L. forbesi mrrp1 expressed as percent identity. (C) Amino acid composition of representatives of the E. scolopes reflectin family. # is the number of times each amino acid occurs in each protein; % is the percent of each amino acid out of the total number of amino acids that occur in each protein.
FIGS. 5a and 5b. FIG. 5a is a ClustalW alignment of E. scolopes reflectins 1a, 2a and 3a and L. forbesi mrrp1 nucleotide sequences (SEQ ID NOs: 1, 5, 11 and 13). These sequences share 62.5% identity. FIG. 5b is a ClustalW alignment of E. scolopes reflectins 1a, 1b, 2a, 2b, 2c, 2d, and 3a nucleotide sequences (SEQ ID NOs: 1, 3, 5, 7, 45 and 9).
FIG. 6. Ribbon structure representing the reflectin 1A gene from E. scolopes. The Reflectin Repeat Peptide (RRP) amino acid sequence used herein is shown below the ribbon structure (SEQ ID NO:15).
FIG. 7. (Left) Circular Dichroism of the RRP showing both near and far-UV absorbance spectra. (Right) X-ray diffraction of RRP dried in a glass capillary. The diffraction pattern is representative of beta-sheet secondary structure.
FIG. 8. (Left) LVTEM image of RRP that had recently been resuspended in water. (Middle) LVTEM image of the same RRP after it was allowed to age for approximately 6 weeks and then spotted on the TEM grid. (Right) AFM topography image and FFT of freshly resuspended RRP.
FIG. 9. (Left and Middle) HVTEM image of aged RRP showing spherical and fibril substructures at different magnifications. (Right) Electron diffraction pattern from the sample on the right. A diffraction aperture was used to limit the field as to only include the proteinaceous material.
FIG. 10. (Left) LVTEM of RRP mixed with ZnSO4. (Middle and Right) HVTEM of RRP mixed AuCl4 at two different magnifications.
FIG. 11. (Left pair) Optical images of precipitated RRP from crystal trials at two different levels of illumination. Similar precipitation occurred in 20% of conditions tested. Images are 3 cm×3 cm. (Right pair) Optical images rotated 30° using a cross-polarized light microscope showing birefringence of the precipitated RRP shown in the left pair of images.
FIG. 12. Recombinant expression of the reflectin protein with (right) and without (left) the N-terminally fused HIS tag. Strong induction of the appropriately sized band on the SDS-page gel can be seen with and without the affinity tag.
FIG. 13 is the nucleotide sequence of the Reflectin 1a sequence using E.coli codon usage (SEQ ID NO:44) to produce the polypeptide sequence of Reflectin 1a (SEQ ID NO:2).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A family of reflectin proteins is described herein; the proteins are deposited in flat, structural platelets in reflective tissues of the squid Euprymna scolopes. These proteins are encoded by at least six genes in three subfamilies and have no reported homologues outside of squids. Reflectins possess 5 repeating domains that are remarkably conserved among members of the family. The proteins have a highly unusual composition with four relatively rare residues (tyrosine, methionine, arginine, and tryptophan) comprising  ̃57% of a reflectin, and several common residues (alanine, isoleucine, leucine, and lysine) occurring in none of the family members. These protein-based reflectors in squids provide a striking example of nanofabrication in animal systems.
Identification of the reflectin proteins associated with reflective tissues of E. scolopes has led to data on amino acid composition and sequence that are unique to electron dense reflective tissues of this species. Analysis of these sequences has shown the composition of the protein to contain a high percentage of arginine, tyrosine, methionine, and tryptophan residues. Within each of the identified reflectin proteins there exists five repeating domains that show strong sequence conservation. Repeat domains have been the hallmark of many structural proteins identified throughout nature, and typically represent the catalytic, or functional, element of the protein. In an effort to elucidate the nature of the reflectin repeat peptides (RRP), the RRP from the third repeat region from the reflectin 1a protein (FIG. 6) was studied, having the sequence MDMSNYMDMYGRYMDRWG (SEQ ID NO:15). The data show that the RRP has reflective activity and also show the secondary structure and properties of the protein in solution and in the presence of metal ions. The data suggest that the RRP can be used in lieu of the whole protein for the same uses.
In vivo, it is believed that the reflecting platelets function by acting as Bragg reflectors with alternating regions of high and low index of refraction materials. The generation of a proteinaceous matrix with a high refractive index is dependent on a number of variables and includes amino acid composition, concentration and crystallinity of the material, and addition of materials such as inorganic metals or in vivo associated ligands/proteins that can complex with the reflectin proteins. In addition, there may exist molecular level organization of the reflectin protein to optimize the overall effective refractive index. These variables are explored through structural and optical characterization of the RRP. Investigation into the discovery of a protein-based reflective material represents a paradigm shift in how structural coloration is viewed. Both the overall bulk materials and microstructure contribute to the reflective ability of these structures, and these mechanisms work cooperatively. While static reflection characterized in this work differs mechanistically from that of dynamic iridophore tissues, the latter most likely derives its function through molecular manipulation of a similar bulk material described herein. The ability to rearrange substructures, alter binding of inorganics and associated proteins, and/or control crystallinity of the bulk can represent some ways in which dynamic reflection can be controlled. It is likely that the overarching principles and structure in both dynamic and static systems are related and should be represented by a conservation of amino acid sequences of the proteins from different species (see also Crookes, et al. 2004 Science Vol. 303, page 235, incorporated by reference in its entirety).
Reflective Tissues
The Hawaiian bobtail squid Euprymna scolopes (Cephalopoda:Sepiolidae; FIG. 1A) is similar to other cephalopod species that have been studied in having both variably reflective tissues, such as the skin of the mantle, and statically reflective tissues, such as those associated with the eye, digestive gland, and light organ. The reflector of the bibbed light organ is a particularly well developed tissue (FIG. 1A-D) that modulates the luminescence produced by a population of the symbiotic bacterium Vibrio fischeri. On each side of the adult light organ, symbiont-containing epithelial tissue comprises a core that is surrounded by the thick silvery reflector. Together with a muscle-derived lens, these dioptrics function to direct the bacterial luminescence ventrally. Consistent with reflectors in other animals, the light organ reflector (LOR) tissue is composed of a thick layer of platelets (FIG. 1C-D).
The Reflectin Proteins
The seven novel reflectin proteins from squid, and their nucleotide sequences are disclosed herein (see the Examples). Thus, some embodiments of the present invention include one or more novel reflectin polypeptides and/or polynucleotides encoding such polypeptides.
In some embodiments, the reflectin polypeptide is at least about 72% identical to at least one of the reflectin proteins from E. scolopes (SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 47), including but not limited to about 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. In further embodiments, the polypeptide is at least about 75% identical to at least one of the reflectin proteins from E. scolopes (SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 47). In further embodiments, the polypeptide is a functional reflectin. In some embodiments, by functional is meant that a polypeptide has the function or activity of a reflectin. In further embodiments, a functional polypeptide shows a golden yellow color upon SDS-PAGE electrophoresis and silver staining. In further embodiments, a functional polypeptide acts as a BRAGG reflector. In further embodiments, a functional polypeptide has a high refractive index. In further embodiments, a functional polypeptide can complex with inorganic metals. In further embodiments a functional polypeptide is active when it has at least one of the above activities and/or qualities.
Further embodiments are polynucleotides that encode the polypeptide recited above. In some embodiments, the polynucleotide is a natural sequence from a squid genome. In further embodiments, the polynucleotide is a derived sequence in which the codon usage for E. coli or an alternative organism is used to express a polypeptide that is at least 72.5% identical to any of SEQ ID NOS:2, 4, 6, 8, 10, 12 and 47. In further embodiments, the polynucleotide is at least about 65% identical to the polynucleotide sequence from E. scolopes reflectin proteins 1a, 1b, 2a, 2b, 2c and 3a (SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 46). In further embodiments, the polynucleotide is at least about 70% identical, including but not limited to: 75%, 77%, 80%, 85%, 90%, 95%, 97.5%, and 99%. In further embodiments, the polynucleotide sequence encodes an active or functional reflectin protein as described above.
As described herein, each reflectin protein is composed of a series of five repeats (designated RRPs). Thus, further embodiments are polypeptides or corresponding polynucleotide sequences that are engineered or truncated in such a way as to remove one or more of the RRPs. Further embodiments are polypeptides or corresponding polynucleotide sequences that have one or more of the RRP sequences removed internally. This can result in an active or functional polypeptide that simply has fewer repeat units but still retains function.
Reflective Repeat Peptide (RRP)
In some embodiments, the RRP is any amino acid sequence corresponding to the motif [α(X)4/5MD(X)5MD(X)3/4], wherein α is MD, FD, or null; X represents any amino acid; the subscripted numbers represent the number of amino acids at that position and the slash represents “or.” For example (X)4/5 means that either four or five amino acids can be at that position, and those four or five amino acids can be any amino acids in any order or combination. Various permutations of the single repeat peptide include SEQ ID NOs: 15-30.
A highly conserved sequence was identified within the RRP motif as shown in Table 1. Thus, in some embodiments, the RRP sequence comprises the motif [MDMQGRY/W](SEQ ID NO: 48). In further embodiments, the RRP comprises any amino acid sequence corresponding to the motif [MDMQGRY/W] (SEQ ID NO: 48) or any variants with conserved amino acids within that sequence, wherein the last amino acid is either Y or W. The sequence may include other amino acids at the N- or C-terminus, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 20 additional residues. In some embodiments, the amino acids at the N- and/or C-terminus are selected from those in SEQ ID NOs: 15-30.







TABLE 1







SEQ ID NO:16







    M S R M T M D F Q G R Y M D S Q G R







SEQ ID NO:17







M D M S G Y Q M D M S G R W M D M Q G R







SEQ ID NO:18







M D M S N Y S M D M Y G R Y M D R W G R







SEQ ID NO:19







M D M S G Y Q M D M Q G R Y M D R W G R







SEQ ID NO:20







F D M S N W Q M D M Q G R W M D N Q G R







SEQ ID NO:21







F G M S N W Q M D M Q G R W M D N Q G R







SEQ ID NO:47







M D Y S N Y Q M D M Q G R Y M D   Q G R







SEQ ID NO:23







M D Y S N W Q M D M Q G R W M D M Q G R







SEQ ID NO:24







M D Y S N Y Q M D M Q G R Y M D M Q G R







SEQ ID NO:25







F D M S N W Q M D M Q G R Y M D Q Y G







SEQ ID NO:26







M D M S N Y S M D M Q G R W M D N Q G R







SEQ ID NO:27







M D M S G Y Q M D M Q G R W M D M Q G R







SEQ ID NO:28







    M S R M T M D F Q G R Y M D R W G R







SEQ ID NO:29







F D M S N W Q M D M Q G R Y M D Q Y G R







SEQ ID NO:30







F D M S R M T M D F Q G R Y M D S Q G R









Because the RRPs are the functional units of the protein, any modifications to the protein sequence that conserve at least one of the RRP sequences can result in an active or functional polypeptide. Likewise, any modifications to a polynucleotide encoding the protein, when such modifications conserve at least one RRP sequence, results in a polynucleotide encoding an active or functional polypeptide. Thus, a further embodiment is any mutated form of the polypeptides and/or polynucleotides that results in at least one conserved RRP within the sequence. Further embodiments include a polypeptide or an encoding polynucleotide, with a sequence that results in a modified RRP that maintains reflective and/or structural characteristics. In some embodiments, permutations and variants any changes that still produce an active reflectin repeat peptide. In some embodiments, the variants still conform to the formula [α(X)4/5MD(X)5MD(X)3/4], wherein α is MD, FD, or null; X represents any amino acid; the subscripted numbers represent the number of amino acids at that position and the slash represents “or.” In a further embodiment, the variants have substantially the sequences of SEQ ID NOs: 15-30 and SEQ ID NO:47 with one or more substitutions, insertions or deletions that conform to either the formula or one of the RRPs described herein or a like amino acid at the equivalent position.
A further embodiment is an RRP that comprises the formula [□(X)4/5MD(X)5MD(X)3/4], including but not limited to SEQ ID NOs: 15-30 as follows:











MSRMTMDFQGRYMDSQGR,
(SEQ ID NO:16)




 



MDMSGYQMDMSGRWMDMQGR,
(SEQ ID NO:17)



 



MDMSNYSMDMYGRYMDRWGR,
(SEQ ID NO:18)



 



MDMSGYQMDMQGRYMDRWGR,
(SEQ ID NO:19)



 



FDMSNWQMDMQGRWMDNQGR,
(SEQ ID NO:20)



 



FGMSNWQMDMQGRWMDNQGR,
(SEQ ID NO:21)



 



MDYSNYQMDMQGRYMDQYG,
(SEQ ID NO:22)



 



MDYSNWQMDMQGRWMDMQGR,
(SEQ ID NO:23)



 



MDYSNYQMDMQGRYMDMQGR,
(SEQ ID NO:24)



 



FDMSNWQMDMQGRYMDQYG,
(SEQ ID NO:25)



 



MDMSNYSMDMQGRWMDNQGR,
(SEQ ID NO:26



 



MDMSGYQMDMQGRWMDMQGR,
(SEQ ID NO:27)



 



MSRMTMDFQGRYMDRWGR,
(SEQ ID NO:28)



 



FDMSNWQMDMQGRYMDQYGR,
(SEQ ID NO:29)



and



 



FDMSRMTMDFQGRYMDSQGR.
(SEQ ID NO:30)






Alternative forms of the RRPs that are still active can be produced using these known sequences and substituting amino acids at equivalent positions or producing chimera of the known peptides. For example, the tryptophan at position 6 of SEQ ID NO:23 can be substituted for the tyrosine at position 6 of SEQ ID NO:22. In addition, any amino acids that have the same properties can be substituted. Further, an amino acid at position 2 can be added to any RRP peptides as long as the peptide still conforms to the formula. Other substitutions can be made as long as they conform generally to the formula and still result in an active polypeptide. In one embodiment, the RRP is MDMSNYMDMYGRYMDRWG (SEQ ID NO:15).
A further embodiment is a polypeptide having, and/or a polynucleotide sequence encoding, one or more of the functional repeat units for the reflectin proteins. In some embodiments, the polypeptide includes 1, 2, 3, or 4 RRPs. The RRPs can be any combination or permutation of those provided in SEQ ID NOS: 15-30 and can contain, for example 2 copies of SEQ ID NO:15, one copy of SEQ ID NO:16 and one copy of SEQ ID NO:17. In some embodiments, the polypeptide has four copies of SEQ ID NO:15.
In some embodiments, the polynucleotide has one or more copies of any combination of the functional repeat units as described above. In some embodiments, the polynucleotide encodes a polypeptide having one or more copies of the functional repeat units. In an alternative embodiment, the polynucleotide results in one or more separately translated RRPs.
Further embodiments include polypeptides and/or polynucleotides having or encoding five copies of the RRPs in a combination or arrangement that is not found in nature. In other words, the repeats are provided in a combination that, while not produced in nature, still results in one or more active polypeptides.
Further embodiments are polypeptides having, and/or polynucleotide sequences encoding, six or more of the functional repeat units for the reflectin proteins. In some embodiments, the polypeptide includes 6 or more RRPs, including but not limited to: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, and 500. In further embodiments, the number of copies is between about 6 and about 30. In further embodiments, the number of copies is more than 6 but less than about 1000. The RRPs can be any combination or permutation of those provided in SEQ ID NOS:15-30 and/or functional modifications thereof, and can contain, for example, 2 copies of SEQ ID NO:15, one copy of SEQ ID NO:16, one copy of SEQ ID NO:17, two copies of SEQ ID NO:18, and one copy of SEQ ID NO:19, and so on. In some embodiments, the polypeptide includes six or more copies of SEQ ID NO:15.
In some embodiments, the polynucleotide includes six or more copies of any combination of the functional repeat units as described above. In some embodiments, the polynucleotide encodes a polypeptide having six or more copies of the functional repeat units. In alternative embodiments, the polynucleotide results in six or more separately translated RRPs.
The sequences between the RRPs can be any sequence that does not negatively affect the secondary or tertiary structure of the RRP and can contain a promoter region, a stop codon, or an initiation codon. Thus, it is to be understood that the polynucleotide for the RRP protein can be expressed as a polyprotein containing two or more RRPs or can be expressed as multiple RRP proteins.
Methods of Expressing Reflectins and/or RRPs
As stated above, the polynucleotide can be expressed as a polyprotein containing one or more RRPs or can be expressed such that each RRP is translated separately and/or transcribed separately. Any promoter can be used that will result in expression in the cell of choice.
In some embodiments, the polynucleotide is provided such that the codon usage for the particular cell results in the polypeptide of choice. For example, the E. coli codon usage can be used to produce the polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 15-30 or any variant thereof.
Methods of Purifying Reflectins and/or RRPs
In some embodiments, the reflectin protein or RRP unit is purified and stored in a buffer having 0.2% or more SDS or an equivalent detergent, including but not limited to: 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%. In further embodiments, the buffer includes SDS at about 0.2 to 2%. In further embodiments, the buffer includes nondetergent sulfobetaine (NDSB) 195, 201, and/or 256 at a concentration of about 0.1 to about 10 M, including but not limited to 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3 M, 2 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, and 0.2 M. In further embodiments the buffer includes CHAPS at a concentration of about 0.1 to about 10%, including but not limited to: 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, and 9.5%. In further embodiments, the buffer contains urea at a concentration of about 3M to about 10M, including but not limited to 4M, 5M, 6M, 7M, 8M, and 9M. In some embodiments the buffer includes DMSO at a concentration of from about 60% to about 100%, including but not limited to 70%, 80%, 90%, and 95%. In further embodiments, the buffer includes trifluoroethanol in a concentration of from about 60% to about 100%, including but not limited to: 70%, 80%, 85%, 90%, 95%, 97.5%, and 99%. In further embodiments, the buffer includes EDTA at a concentration of from about 0.2 M to about 3M, including but not limited to 0.3M, 0.4 M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.5M, 1.75M, 2M, 2.5M, and 2.75M. In further embodiments, the buffer includes ammonium acetate and/or ammonium sulfate in a concentration of about 0.2M to about 3M, including but not limited to 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.5M, 1.75M, 2M, 2.5M, and 2.75M
In further embodiments, the buffer contains one or more metal ions added at a concentration of about 10 mM or less, to about 100 mM or more, including but not limited to: 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, and 90 mM. In some embodiments, the metal ions are zinc and/or gold, including but not limited to ZnSO4 and AuCl4.
Methods of Using Reflectins and/or RRPs
Any methods of using proteins that reflect and/or have a high refractive index can be used. Some methods include, but are not limited to: as a reporter protein to characterize transcription of a protein and/or a promoter, in nanostructured supramolecular devices, and for nanofabrication of any type of material, for example reflective biomaterials.
EXAMPLES
In Examples 1-3, a total of 6 different reflectin proteins from the Squid E. scolopes were identified and sequenced. Example 4 provides an analysis of the sequence. In Examples 5-12, the smallest active portion of the protein, the Reflectin Repeat Peptide (RRP) is expressed, identified and characterized. In Examples 13-14 various methods for the use of the peptides and proteins are provided.
The specimens of E. scolopes were obtained from the shallow reef flats of Oahu, Hi., transported to circulating natural seawater aquaria at the University of Hawaii, and maintained as described in Weis, et al. Biol. Bull. 1993, 184, p. 309 (herein incorporated by reference in its entirety). All chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) unless otherwise noted.
Example 1 
Isolation of Reflectin Proteins from E. scolopes 
To enrich for the reflecting, the light organ reflector (LOR) was first homogenized in 50 mM sodium phosphate buffer, pH 7.4, with 0.1 M NaCl (PBS) in a ground glass homogenizer on ice to extract the aqueous soluble fraction. The total homogenate was centrifuged at 20,800×g for 15 min at 4° C. The resulting supernatant was removed. The pelleted material was then washed by repeatedly resuspending it in PBS and centrifuging the resuspension at 20,800×g for 15 min at 4° C. to re-pellet the aqueous-insoluble material. The resulting washed pellet was resuspended in 2% SDS in PBS to extract the SDS-soluble fraction that contained the reflecting. The suspension was then centrifuged, as described above, and the supernatant retained for analyses.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a Bio-Rad Mini-Protean II electrophoresis system (Bio-Rad, Hercules, Calif.) under standard SDS-PAGE procedures (modified from Laemmli, Nature 227, p. 680). The protein concentrations of all fractions were determined spectrophotometrically.
Extractions of total protein from the E. scolopes LOR revealed a set of three abundant polypeptides that resolved between 33-36 kDa on SDS-PAGE and were a characteristic golden-yellow color upon silver staining (FIG. 1E, E′). These polypeptides (reflecting) were not detected in the aqueous soluble fraction of the LOR, but were abundant in the supernatant of the SDS-solubilized pellet, composing  ̃40% of the proteinaceous component of the LOR.
To determine the approximate concentrations of reflectins in the LOR, the protein concentration of the whole homogenate, PBS-soluble supernatant, and the SDS-soluble fraction of the light organ reflector were each determined. From SDS-PAGE analysis of the SDS-soluble fraction of the LOR, reflectins made up at least about 50% of the total protein in this fraction. This value was used to back-calculate to determine what proportion of the whole homogenate was reflecting.
Example 2 
Tissue Localization of Reflectins
To localize the reflecting within the LOR, polyclonal antibodies were generated against gel-purified reflectin proteins (FIG. 1E, lane 5) and used in immunocytochemical and immunoblot analyses.
To generate material for antibody production, the reflectin proteins were purified directly from the SDS-PAGE gel of the LOR proteins. Briefly, the 2% SDS-soluble fraction of light organ reflectors from seven adult animals was applied to a stacking gel without wells. The portion of the gel containing the reflectins was excised from the gel and homogenized in 2% SDS in PBS. The resulting slurry was transferred to a Microcon-100 spin filter (Millipore Corp., Bedford, Mass.) and centrifuged at 3000×g for 10 min at room temperature. An aliquot of the filtrate was resolved on SDS-PAGE, that indicated that the desired proteins had been isolated. The remaining filtrate protein was used in the production of polyclonal antibodies (Covance Research Products, Inc., Denver, Pa.). For western blot analysis, protein extractions of the SDS-soluble pellet of various adult tissues were performed as described above for the LOR. The protein concentration of each extraction was determined spectrophotometrically and SDS-PAGE gels were run according to standard protocol. The companion SDS-PAGE gel was silver stained according to standard procedures. For western blots, proteins were electrophoretically transferred to nitrocellulose membrane (Bio-Rad, Hercules, Calif.). Blots were blocked overnight in 4% milk in 50 mM Tris, 150 mM NaCl, 0.5% Tween 20, pH 7.5 (TTBS). Following this blocking step, blots were incubated for 1 h in a 1:10,000 dilution of antiserum in 1% milk/TTBS. Blots were washed 3 times in 1% milk/TTBS and then incubated for 45 min in a mixture of 1:3000 goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Bio-Rad, Hercules, Calif.) and 1:3300 avidin-conjugated horseradish peroxidase to detect biotinylated molecular mass markers (Bio-Rad, Hercules, Calif.). These detection reagents were diluted in 1% milk/TTBS. Detection of cross-reactive bands was achieved by chemiluminescence (ECL chemiluminescence kit, Amersham Biosciences Corp, Piscataway, N.J.).
Immunogold localization by transmission electron microscopy (TEM) was performed as follows: Light microscopy and TEM of squid tissues were performed as described in McFall-Ngai, et al. (Biol. Bull., 1990, 184, p. 296) and Montgomery et al. (J. Biol. Chem., 1992, 267, p. 20999), each of which is herein incorporated by reference in its entirety. Immunocytochemistry at the TEM level was also performed as described in Weis et al (see Example 1) except that anti-reflectin antibodies were used as the primary antibody at a 1:1000 dilution. A 1:50 dilution of goat anti-rabbit IgG conjugated to 15-nm gold spheres (Ted Pella, Redding, Calif.) was used as the secondary antibody. To control for nonspecific binding of the secondary antibody, a subset of the grids was incubated with a 1:1000 dilution of preimmune serum. TEM was performed on a JEOL 100 CX transmission electron microscope at the University of Southern California (Los Angeles, Calif.).
The reflectin antibodies strongly recognized the LOR, but not the bacteria-containing epithelium, the ink sac, or lens of the light organ (FIG. 2A). The results of the immunogold localization showed that the reflectins in the LOR were directly associated with the LOR platelets (FIG. 2A′) and that no labeling was detected in the surrounding connective tissue or ink granules (FIG. 2A′). Higher magnification TEM images of the LOR demonstrated that the antibodies cross reacted specifically with the electron-dense platelets but not the inter-platelet region (FIG. 2A′, inset).
Proteins with similar molecular mass, biochemistry, and antigenicity to LOR reflectins were found in all reflective tissues of E. scolopes. Silver-stained SDS-PAGE gels revealed that the characteristic golden-yellow bands at 33-36 kDa were detectable in reflective tissues. In addition, these proteins cross reacted with antibodies to LOR reflectins in western blot analyses (FIGS. 1A, 2B). Cross-reactive bands were also found at other molecular weights (26, 55, and 120 kDa), and corresponding golden-yellow bands were present on the SDS-PAGE gels of these tissues. Because the antibodies were generated from a very discrete region of a gel, and these other bands only occur in reflective tissues, it is likely that the antibodies were cross reacting with other members of the reflectin family or closely related proteins. Cross reactivity was low or undetectable in non-reflective tissues (gills, muscle, eye lens, and light organ lens) (FIG. 2B).
Example 3 
Sequencing and Analysis of Reflectins Genes
The sequences of three tryptic peptides (FIG. 3A) from tryptic digestion of gel-purified reflectins (FIG. 1E, lane 5) were used to identify reflectin cDNAs from predicted translations of E. scolopes cDNA and EST library clones as described below. The tryptic peptides had ambiguities in them as expressed by an “X” in the following sequences: SMFNYGWMMDGDR (SEQ ID NO:31), EGYYPNYSYGR (SEQ ID NO:32), and YFDMSNWQMDMQGR (SEQ ID NO:33).
Protein extracts from light organs were subjected to SDS-PAGE. Reflectin bands were excised from the gel and subjected to trypsin digestion. The resulting tryptic peptides were sequenced by mass spectrometry (Harvard Microchemistry Facility, Cambridge, Mass.). The amino acid sequences of three tryptic peptides were used to screen predicted translations of sequences from cDNA pools constructed from the light organs of juvenile animals (SEQ ID NOs: 31-33). One partial sequence was obtained from this pool, the translation of which contained 2 of the 3 tryptic peptide sequences. This small tryptic peptide sequence had significant similarity (88%) to Loligo forbesi ‘methionine-rich repeat protein 1’ (mrrp1; accession no. CAC86921) (SEQ ID NO:14).
Using the nucleotide sequence of L. forbesi mrrp1 (SEQ ID NO:13) for information about possible length and the sequence of the E. scolopes partial clone for primer design (Table 2), RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) was conducted on an E. scolopes light organ cDNA pool to obtain full-length clones. 5′ and 3′ RACE-PCRs were performed on the clone using the SMART RACE cDNA amplification kit (BD Biosciences Clontech, Palo Alto, Calif.) and primers specific to the cDNA clone (see Table 2; 33F3, 33R2, 33R3, 33R4). First-strand synthesis was performed on 195 ng of light organ mRNA according to the manufacturer's instructions. Both 5′ and 3′ RACE reaction conditions were as follows: 5 cycles of 94° C. for 30 sec, 72° C. for 3 min; 5 cycles of 94° C. for 30 sec, 70° C. for 30 sec, 72° C. for 3 min; 25 cycles of 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 3 min. RACE products were run on 1% agarose gels and stained with ethidium bromide according to standard procedures. RACE products were gel-extracted (GeneClean kit, Bio101, Carlsbad, Calif.) and ligated into the pGEM-T easy vector (Promega Corp., Madison, Wis.). Products from the ligation reactions were transformed into E. coli DH5alpha and transformants were screened for inserts by blue-white screening on LB-carbenicillin (50 μg/ml) plates containing 0.9 mg IPTG and 800 μg X-gal (Promega Corp, Madison, Wis.). White colonies were further screened by restriction enzyme digestion (EcoRI) to identify those transformants with plasmids that contained appropriately sized inserts. Plasmids from positive colonies were mini-prepped (Qiagen Inc., Valencia, Calif.) and sequenced at the University of Hawaii Biotechnology/Molecular Biology Instrumentation and Training Facility.








TABLE 2







Primers used for RACE-PCR and standard PCR reactions.











SEQ




ID












Primer
Primer type
Direction
Sequence (5′ to 3′)
NO:















33F3
3′ RACE
forward
CGC CAC TGC AAC CCG TAT AGC CAA TGG
34



 


33R2
5′ RACE
reverse
CCA ATA GGG GCT GCA GTA GCG TCC
35


 


33R3
5′ RACE
reverse
GTT GCC GGA GCG GTT CCA GTG GTT GTA A
36


 


33R4
5′ RACE
reverse
CCC GGG GTA GTT CCA GTA TCT GCC AT
37


 


33AF
standard PCR
forward
ATG AAC CGT TTT ATG AAC AGA TAC CG
38


 


33BF
standard PCR
reverse
ATG AAC CGT TAC ATG AAC CGA TTC CG
39


 


33A1R
standard PCR
reverse
GTA ATA GTC GTT CAT TCC GTA TTG GTC C
40


 


33B1R
standard PCR
reverse
GAG CAA GAC GTT CAA GAA TTT CAG ACG
41


 


33B2R
standard PCR
reverse
CCA GTT GTA ATA ATT ATA GGG ATA ATC C
42


 


33BR
standard PCR
reverse
CCA TGT ATC GTC CCT GCA TGT CCA TCC
43









Genomic DNA extracted from the light organ of a single adult E. scolopes was used as a template for PCR reactions to: i) determine whether genes for all 6 cDNAs occur in the genome, or are the result of alternative splicing or allelic differences; and, ii) provide information about gene structure. To amplify reflectin genes from genomic DNA, PCR reactions were carried out using all possible combinations of 2 forward primers: 33AF(SEQ ID NO:38), or 33BF(SEQ ID NO:39) and 4 reverse primers: 33A1 R (SEQ ID NO:40), 33B1R(SEQ ID NO:41), 33B2R(SEQ ID NO:42), and 33BR(SEQ ID NO:43); (see Table 2). Reactions were carried out with 1.5 mM MgCl2, 1 μM each forward and reverse primers, 1 mM dNTPs, and 2.5 U Taq DNA polymerase (Promega Corp., Madison, Wis.). Reaction conditions were as follows: 94° C. for 2 min; 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1.5 min for 35 cycles; 72° C. for 5 min. PCR products were cloned and sequenced as outlined above for RACE products.
In addition to reflectin gene sequences obtained from light organ cDNA and genomic DNA, sequences were obtained from an EST database being constructed from light organ cDNA libraries of juvenile animals.
All six full-length reflectin sequences contained stop codons followed by polyadenylated tails, that demonstrates that there was no genomic DNA contamination. Accession numbers for the reflectins can be found in Table 3.
To produce the cDNA pool, RNA was isolated from the light organs from juvenile E. scolopes that were dissected, placed in RNAlater (Ambion, Inc., Austin, Tex.), and stored at −20° C. Total RNA was extracted as follows: 80 juvenile light organs were homogenized in 600 μl TriPure Isolation Reagent (Roche Applied Sciences, Indianapolis, Ind.) for 30 min on ice in a ground glass homogenizer. The homogenate was incubated for 5 min at room temperature, and then 120 μl of chloroform was added to the homogenate. The mixture was allowed to stand for an additional 10 min at room temperature, and then centrifuged at 12,000×g for 15 min at 4° C. The upper aqueous phase was transferred to a new tube and 300 μl of isopropanol was added. This mixture was incubated for 7 min at room temperature to allow precipitation of total RNA and then centrifuged at 10,800×g for 10 min at 4° C. The supernatant was discarded and the RNA pellet was washed once with 75% ethanol. The pellet was air dried and was then resuspended in 50 μl of RNase-free water. The resuspension was then incubated at 55° C. for 15 min and assessed for quantity and purity spectrophotometrically. mRNA was extracted from total RNA using the MPG mRNA Purification Kit (CPG, Lincoln Park, N.J.) according to the manufacturer's instructions. mRNA was quantified, assessed for purity spectrophotometrically, and resolved on a 1% agarose gel to confirm that the RNA was not degraded.
Genomic DNA was extracted from one adult light organ reflector using the portion of the MasterPure Complete DNA and RNA Purification Kit (Epicentre, Madison, Wis.) designed to isolated the DNA. During the extraction, the sample was treated with 5 μg RNase A to digest single-stranded RNAs. The DNA was quantified and assessed for purity spectrophotometrically.







TABLE 3







Comparison of predicted protein characteristics of reflectins


1a-3a and L. forbesi mrrp1 (Lf).













Accession

molecular

SEQ ID



number
# residues
mass (kDa)
pI
NO
















1a
AY294649
283
36.7
8.84
2


1b
AY294650
282
36.2
8.82
4


2a
AY294652
283
36.7
8.84
6


2b
AY294653
284
37.0
8.80
8


2c
AY294654
284
37.2
8.81
10


3a
AY294651
288
37.6
8.81
12


Lf
CAC86921
264
32.8
7.61
14





# residues, number of total amino acids;


pI, predicted isoelectric point.






Example 4 
Analysis of Reflectin Sequences and Homologies
RACE-PCR conducted on light organ cDNA pools using reflectin primers (Table 2) identified six similar reflectin cDNAs (FIG. 1; Table 3), suggesting that several genes encoding reflecting were expressed in the light organ. Sequencing of PCR products from the amplification of genomic DNA provided evidence that all six reflectin genes amplified from the cDNA pools are represented in the genome of a single individual. None of the reflectin genes amplified from genomic DNA possessed introns. Only one entry in the nucleotide databases had similarity to the E. scolopes reflecting, a gene sequence from the European squid Loligo forbesi (Weiss et al. NCBI accession number CAC86921, 2002) that encodes ‘methionine-rich repeat protein 1’ (FIG. 1, A and B; Table 3) (accession no. CAC86921). However, there was no known function for L. forbesi mrrp 1, and this protein was less than 72.5% identical to any of the E. scolopes sequences, while the reflecting from E. scolopes were between 85 and 98% identical to each other (see FIG. 4B).
The derived amino acid sequences of the six full-length clones were aligned, demonstrating that the reflecting are highly similar (85.0-98.6%) and group into three subfamilies (FIG. 4A and B; Table 3). Analysis of their structure revealed some unusual characteristics. Reflectins possess a highly unusual amino acid composition (FIG. 4C); six amino acids (Y, M, R, N, G, D) compose over 70% of the total, and four other amino acids (A, I, L, K) are absent. Although their SDS solubility suggested that they can be membrane associated, further analyses demonstrated that they are not predicted to possess hydrophobic or charged clusters, transmembrane domains, or glycosylphosphatidylinositol anchors. However, each reflectin is composed of five repeating domains (FIG. 4). When these repeats are aligned (FIG. 3A, lower), a ‘core’ subdomain (SD) of 18-20 amino acids is revealed; the subdomains were defined by the presence of a repeating motif [α(X)4/5MD(X)5MD(X)3/4]. that occurs in 4 of the 5 subdomains. In this motif, X represents any amino acid; the subscripted numbers represent the number of amino acids at that position and the slash represents “or.” In these subdomains 21 of 23 methionine residues occur in the same relative position in the repeat. The subdomains are enriched in M, R, G, D, S, and Q, and depleted in Y, N, W, P, and F relative to the whole protein (Table 4). Inter- and intra-protein subdomain alignments demonstrated that individual subdomains from different reflecting (e.g., SD1 from 1a vs. SD1 from 2a) are more similar to each other (80-100%) than are different subdomains (e.g., SD1 from 1a vs. SD2 from 1a) of the same reflectin (55-70%) (FIG. 4B). These data suggest greater functional constraint on the sequence within a subdomain across the family.







TABLE 4







Amino acid composition of the subdomains of reflectins and


comparison to amino acid composition of the complete protein.


Reflectin 1a was used as a representative protein.













outside





in
sub-

depleted (−) or


amino
subdomains
domains
in total protein
enriched (+) in














acid
#
%
#
%
#
%
subdomains

















Y
9
9.2
50
27.0
59
19.8
−


M
23
23.5
19
10.3
42
14.6
+


R
22
22.4
11
5.9
33
11.8
+


N
4
4.1
24
13.0
28
9.7
−


G
11
11.2
13
7.0
24
8.3
+


D
17
17.3
6
3.2
23
8.3
+


W
4
4.1
7
3.8
11
5.9
−


S
7
7.1
8
4.3
15
4.9
+


P
0
0.0
13
7.0
13
4.5
−


Q
10
10.2
3
1.6
13
4.5
+


F
2
2.0
7
3.8
9
2.8
−


E
0
0.0
4
2.2
4
1.4
−


H
0
0.0
4
2.2
4
1.4
−


C
0
0.0
4
2.2
4
1.4
−


T
0
0.0
3
1.6
3
0.3
−


V
0
0.0
1
0.5
1
0.3
−


A
0
0.0
0
0
0
0.0
na


I
0
0.0
0
0
0
0.0
na


L
0
0.0
0
0
0
0.0
na


K
0
0.0
0
0
0
0.0
na





in subdomains, amino acid representation within all 5 subdomains (n = 98 amino acids);


outside subdomains, amino acid representation outside all 5 subdomains (n = 185 amino acids);


in total protein, representation of each amino acid in the entire sequence of the protein (n = 283 amino acids);


#, occurrence of each amino acid;


%, percent occurrence of each amino acid;


na, not applicable.






The derived amino acid sequences of the six full-length reflectin clones were aligned, and all three tryptic peptides from reflectin protein sequencing were found in the translation of all clones (FIG. 4A underlined sequences). The alignment demonstrates that the proteins are very similar to one another as well as to the L. forbesi mrrp1 (FIG. 4A). Pairwise comparisons of all six sequences suggests a grouping of the E. scolopes reflectins into three subfamilies, reflectins 1, 2 and 3 (FIG. 4A and B), based on percentage identity of the amino acids and the positions of deletions/insertions.
As shown in FIG. 5, alignment of the polynucleotide sequences results in considerably less identity. For example, E. scolopes reflectins 1a, 2a, 3a, and L. forbesi share only 63.5% identity. Identity between the E. scolopes proteins is better than with L. forbesi, resulting in about 77.2% identity.
The E. scolopes reflectins have theoretical masses between 36.2 and 37.6 kDa, predicted isoelectric points between 8.80-8.84 (Table 3), and a highly unusual amino acid composition (FIG. 4C). Comparing the reflectins with other proteins by available protein analysis algorithms revealed that the reflectins possess extremely high usage of Y, M, and R, a high usage of W, and an extremely low usage of T, V, A, I, L, and K. The reflectins possess one of the highest tyrosine contents (19.8%) among current Protein Database proteins. Other tyrosine-rich proteins include an assortment of extracellular matrix and structural proteins including insect storage proteins (11-27%), keratin-associated proteins (12-17%), dental enamel peptides (13%), tyrosine-rich acidic matrix proteins (10%), and glycoproteins involved in oocyst cell wall formation in apicomplexan parasites (10%), suggesting that this amino acid may be important in the formation of protein superstructures.
Application of algorithms that predict secondary structure revealed high representation of order-promoting residues (W, Y, F, and N), the absence of 4 residues (A, I, L, and K) that are abundant in other proteins and often used in packing of hydrophobic cores, and the presence of highly ordered repeats suggest that the reflectins fold and pack in unusual ways.
Analysis of reflectin clones was carried out using MacVector 7.1.1 (Accelrys, San Diego, Calif.). Sequence alignments were performed by ClustalV. Amino acid sequence analysis was performed using the following programs available on the ExPASy Molecular Biology server (www.ExPASy.org): SignalP, PredictProtein, TMHMM, ProtParam, Radar, SAPS, nnPredict, Jpred, Sulfinator, and big-PI Predictor.
The identification and characterization of the reflectins confirmed that while the majority of animal reflective tissues are composed of purine platelets, cephalopod reflector platelets are proteinaceous. Reflectins, a previously undescribed protein family with skewed amino acid compositions, repeating domains, and localized deposition, are thus far restricted to cephalopods. They represent a striking example of natural nanofabrication of photonic structures in these animals.
Interestingly, a seventh reflectin protein sequence was identified and called reflectin 2d (SEQ ID NOs: 45 and 46) because of its homology to the other reflectin 2 proteins. Reflectin 2d was amplified from genomic DNA but has not yet been identified in the light organ cDNA pool. On an amino acid level, it is 89.7% identical to 1a, 86.4% identical to 1b, 98.9% identical to 2c, 96.0% identical to 3a and 70.5% identical to L. forbesi mrrp. This reflectin appears to have resulted from genomic amplification and has not yet been shown to be expressed in the LOR.
To further analyze the polypeptides and to determine the minimum functional or active portion, an RRP was prepared as described below and analyzed.
Example 5 
Structural Characterization of the Reflective Repeat Peptide From the Reflectin 1A Gene
The 18 amino acid synthetic RRP (New England Peptide) shown in FIG. 6 (SEQ ID NO:15) was resuspended in water to a final concentration of 10 mg/ml. Secondary structure determination was undertaken using a combination of Circular Dichroism (CD) and X-ray Diffraction (FIG. 7). The CD spectrum of the suspension revealed a mostly beta-sheet character with peak absorbance at 207 nm in the far-UV. The CD spectrum of protein in the near-UV (250-350 nm) can be sensitive to certain aspects of tertiary structure. At these wavelengths the chromophores are the aromatic amino acids and disulfide bonds, and the CD signals they produce can represent a defined tertiary structure of the protein. Signals in this region are attributed to tyrosine (270-290 mn), tryptophan (280-300 nm), and disulfide bonds that give rise to a broad but weak signal throughout the near-UV spectrum. The presence of a strong near-UV signal in the CD spectrum for the RRP was an indication that the protein was folded into a well-defined orientation with a defined tertiary structure in solution. To complement the CD data, the resuspended solution was dried in a glass capillary for X-ray diffraction analysis. After drying, the protein remained optically clear and gave rise to distinct rings in the X-ray diffraction pattern at 0.38, 0.46, and 1.15 nm, indicative of a crystalline beta-sheet structure. The fact that the peptide maintained its overall secondary structure following the drying process was due to the stability of the peptide interactions.
Example 6 
LVTEM Analysis
In order to determine the contribution of the peptide in higher-ordered tertiary and quaternary structures, electron microscope studies of the peptide spotted onto copper grids with an amorphous carbon support were performed. A combination of Low-voltage transmission electron microscopy (LVTEM) and High-voltage transmission electron microscopy (HVTEM) was used to characterize the peptide. Low-voltage electron microscopy allowed for imaging the protein materials without staining and prevented beam damage to otherwise delicate structures. LVTEM micrographs of newly resuspended RRP and peptide that was allowed to sit for several weeks after resuspension revealed a dramatic difference in the overall crystallinity and structure of the material. FIG. 8 shows the LVTEM images of the two samples. The newly resuspended RRP showed distinct strands and small spheres. The imaged strands were  ̃8 nm in diameter and the small spheres measure  ̃10 nm in diameter. LVTEM of the aged sample revealed a dramatically more electron dense sample and only spherical structures with diameters of  ̃12 nm were resolved from the image. The structure of the more electron dense regions was not resolved due to the nature of the lower electron voltage used by the microscope. Corroboration for the filamentous nature of newly resuspended RRP was obtained using Atomic Force Microscopy (AFM). The RRP was spotted onto a silicon wafer and allowed to dry in air. The AFM image showed long aligned fibrils that extended for several microns (FIG. 8). A Fast Fourier-Transformed (FFT) image of the AFM topographic image revealed that the spacing between crystals was regular and equal to 8 nm on average, revealing periodicity of the native RRP protein.
Example 7 
Solubility Of Reflectins
To determine the relative solubility of reflectins, insoluble proteins were extracted from Euprymna scolopes light organ reflector (LOR) or eye reflectors by homogenization in 50 mM sodium phosphate buffer, pH 7.4, with 0.1 M NaCl (PBS) on ice. The total homogenate was centrifuged at 20,800×g for 15 min at 4° C. The pelleted insoluble material was then washed repeatedly in PBS. After the final wash, the material was resuspended in PBS. The resuspension was aliquotted to individual tubes. Each tube was centrifuged at 20,800×g for 15 min at 4° C. to re-pellet the insoluble material. The supernatant was discarded. The resulting pellet was resuspended in 25 μl of treatment solution and centrifuged again. The supernatant was removed, mixed with SDS-PAGE buffer, boiled for 5 min, and subjected to SDS-PAGE. Relative solubility in the presence of various reagents (Table 5) was assessed by comparison with a control sample that had been solubilized in 2% SDS.







TABLE 5







Solubility of reflectins in various reagents.











relative solubility



Treatment
of reflectins







0.1% SDS
−



0.2% SDS
++



1-2% SDS
+++++



1% Triton X-100
−



0.5% Tween-20
−



1% CHAPS
−



1M nondetergent sulfobetaine (NDSB)-195
+



1M NDSB-195 + 2% CHAPS
+



1M NDSB-201
++



1M NDSB-201 + 2% CHAPS
+



1M NDSB-256
+



1M NDSB-256 + 2% CHAPS
−



1 M urea
−



3 M urea
−



3 M urea, 0.3 M NaCl
+



8 M urea
−



8 M urea, 2% CHAPS
++



50% dimethylsulfoxide
−



100% dimethylsulfoxide
++++



100% trifluoroethanol
++



100% acetonitrile
−



0.5 M EDTA
+



1 M ammonium acetate
++



1 M ammonium sulfate
++



1 M lithium chloride
+



5 M lithium chloride
+



2 M sodium chloride in PBS, pH 7.4
+



3 M sodium chloride in PBS, pH 7.4
+



0.25 M sodium sulfate, pH 6
−



0.25 M ammonium carbonate, pH 8.5
−



0.1 M sodium carbonate, pH 11
+










This information can be used in combination with the information gained in other Examples herein to identify the best conditions for the use, native conformation, reflectivity and solubilization of the RRP proteins.
Example 8 
HVTEM Analysis
A more detailed inspection into the molecular structure was undertaken using HVTEM. Newly resuspended RRP was largely unstable in the high-voltage beam and a high-resolution image was not obtainable. The aged peptide, with higher electron density in the LVTEM beam, was significantly more stable in the higher voltage beam and an underlying high-resolution structure was obtained. FIG. 9 shows these high-resolution images at two different magnifications. Within the RRP structure, there existed both the spherical and fibril structures noted in the LVTEM study, albeit with a tighter association between substructures. The interaction between these two structures lead to a large thread-like formation that could span between 30 and 200 nm. Due to the increased beam stability, a distinct and repeatable electron diffraction pattern was resolved and is included in FIG. 9.
Incorporation of inorganic metals to the native reflectin based platelets was theorized to provide structural stability and/or an increase in overall effective refractive index of the material. This was explored in Example 9.
Example 9 
Interaction with Metals
Ultra-thin sections of the reflectin platelets are obtained. HRTEM and elemental analysis of these tissues reveals whether inorganic metals are a necessary inclusion for protein stability and contribute to the effective refractive index of the material.
Due to the unique amino acid composition of the RRP, certain inorganic metals can be used to affect their structure and effective refractive index. 50 mM ZnSO4 was added to the RRP and spotted on a copper grid for LVTEM analysis. Zn addition altered the overall structure of the RRP as seen in FIG. 10. The fibrils following Zn addition had a slightly wider diameter of  ̃15 mn and seemed to possess a greater rigidity. Zn is known to be important in control of both tertiary and quaternary structure of proteins and this result suggests that it plays a role in the structure of the RRP. Tyrosine residues, which are found in a high percentage of both the RRP and the full length reflectin proteins, have been demonstrated previously to potentiate the reduction of AuCl4 to metallic gold with a high degree of crystallinity. Following AuCl4 addition to RRP, there was an overnight precipitation of the RRP in solution. LVTEM and HVTEM images of the RRP-Au precipitate were analyzed. HRTEM images showed that the gold was able to incorporate within the microstructural framework of the RRP. Spherical gold nanoparticles were embedded within the proteinaceous material and had similar dimensions ( ̃12 nm) of the spherical structures observed for aged RRP (FIG. 9). Composition of the nanoparticles were confirmed to be crystalline Au0 by indexing the refraction pattern from these regions and measuring the lattice spacing of the nanoparticles from the high resolution image. In addition, composition of the electron-dense threads was confirmed to be a composite inorganic-organic material of RRP and Au from energy dispersive spectroscopy. The ability to incorporate inorganic metals by using the RRP as a template showed that this approach can be used to control index of refraction of the composite material and tune the material appropriately.
Bragg reflection from native platelets is hypothesized to be accomplished through the use of a high index of refraction protein material. The unique amino acid contribution of the reflectin proteins to include the aromatic residues tyrosine and tryptophan is likely to contribute to the overall bulk refractive index. The use of inorganics to increase the bulk refractive index was also discussed above. While the bulk characteristics are integral in the reflection process, it is likely that there exists a high degree of crystallinity of the protein and/or protein-inorganic matrix and is necessary in the reflection mechanism. Protein concentrations within the reflective organelles would have to be extremely high to produce the necessary refractive index mismatch with the outlying cellular components. Furthermore, if the protein bulk was generally amorphous, there would exist a high level of scatter within the reflectin organelle and reduce the overall reflectivity of the platelets. To circumvent this problem, the production of protein matrix with a high degree of crystallinity would generate a material of extremely high protein concentration with little scatter and high reflection. To determine conditions necessary for crystallization of the RRP, a number of different conditions were explored using a hanging-drop vapor diffusion protein crystallization technique. This combinatorial approach showed that in almost 20% of the conditions, the RRP precipitated out of solution within a day. Within the precipitate, the RRP appeared to possess clear regions that appeared reflective under an optical microscope with overhead illumination as seen in FIG. 11. These samples were transferred to a glass slide and viewed under a cross-polarizing microscope to determine birefringence of the material. Indeed, the material demonstrated significant birefringence as associated with ordered materials. Reversible crystallization can lead to the development of a tunable Bragg reflecting system by controlling the levels of scattered and reflected light.
The characterizations from Examples 6-9 show that the RRP sequence has the potential for mineralization/metallization of inorganics and that certain inorganics can be involved in folding (tertiary structure) of the peptide and/or the effective refractive index.
Example 10 
Production of a Bacterial Expression System for Reflectin 1A
In order to explore the full-length protein complex and produce large quantities of the protein, a reflectin protein that was based on the amino acid sequence from reflectin 1a was recombinantly expressed (SEQ ID NOs: 1 and 2). A synthetic gene based on this sequence was produced because of the difficulty associated with polymerase chain reaction amplification of proteins with repetitive sequences and the large arginine content of the sequence. Because of the codon bias for E. coli, DNA sequences possessing rare codons not used by this organism are not effectively recombinantly expressed. The sequence was optimized so that it best reflected the codons used natively by E. coli. Initial recombinant experiments have shown that the reflectin 1a sequence can be expressed using an IPTG induced BL21 system. SEQ ID NOS: 44 and 45 provide the nucleotide sequence of these constructs. FIG. 12 shows the expression of this sequence and one that contains a N-terminal hexahistidine fusion to aid in subsequent purification (SEQ ID NO:45). Although in many buffers the protein is predominantly insoluble, the analyses disclosed herein permit selection of buffer components that can facilitate refolding of the recombinant protein into its native conformation.
Example 11 
Use of Reflectins for Nanofabrication
Protein-based nanofabrication is a frontier area in biomimetics, in which protein structures are engineered to be used as biomaterials(Zhang, et al. Curr. Opin. Chem. Biol. 2002, Vol. 6, page 865, herein incorporated by reference in its entirety). For example, numerous biomaterials can be either genetically altered to produce reflectin and/or RRP proteins, including but not limited to wood, silk, cotton, flax and burlap. Alternatively, the purified reflectin and/or RRP protein can be admixed with a synthetic material to produce a semi-biomaterial, including but not limited to: polyester, metals, plastics, and the like.
Example 12 
Use of Reflectins for Nanostructured Supramolecular Devices
Future embodiments of the invention provide reflectins that can support the ‘bottom-up’ synthesis of nanostructured supramolecular devices, especially those used in spectroscopic and optic applications (Vukusic, et al. Nature 2003, vol. 424, page 852, herein incorporated by reference in its entirety). For example, reflectin-based nanoreflectors can be coupled with artificial photosynthetic membranes (Steinberg-Yfach, et al., Nature 1997, Vol. 392, page 479, herein incorporated by reference in its entirety) or with bacteriorhodopsin-based bioelectronic devices (Wise, et al. Trends Biotechnol. 2002, Vol. 20, page 387, herein incorporated by reference in its entirety) to enhance the power and efficiency of these systems.
Example 13 
Use as a Reporter Gene
Currently, two of the most common reporter genes for use in transcriptional studies are green fluorescent protein (GFP) and β galactosidase. Because of their unique qualities, any of the reflectin proteins or RRPs can be used in addition to these reporter genes or as an alternative. The RRP proteins are operably linked to any promoter known to one of skill in the art that is being studied. The expression of the promoter is analyzed with respect to the amount of RRP produced as measured by spectroscopy, UV absorption, or microscopy in the presence of various filters.
The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein.
Furthermore, the skilled artisan will recognize the interchangeability of various features from different embodiments. Similarly, the various features and steps discussed above, as well as other known equivalents for each such feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with principles described herein.
Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosures of preferred embodiments herein, but instead by reference to claims attached hereto.

## Claims

1. A biomimetic reflective material comprising a first component, the first component comprising at least one polypeptide selected from the group consisting of:
(a) a reflectin polypeptide having at least 87.2% identity to SEQ ID NO:2;
(b) a polypeptide having at least one and not more than four repeat units of the reflectin polypeptide;
(c) a polypeptide comprising at least six repeat units of the reflectin polypeptide; and
(d) any combination of (a) through (c); the first component being in combination with at least a second component compatible with the first component, such that the combination forms a biomimetic reflective material.

2. The biomimetic reflective material of claim 1, the material comprising a metal ion.

3. The biomimetic reflective material of claim 1, the material having at least a first and a second refractive state, wherein the material in the first refractive state has a refractive index that is different from a refractive index of the material in the second state.

4. A method of producing a biomimetic reflective material comprising providing a first component comprising at least one polypeptide selected from the group consisting of:
(a) a reflectin polypeptide having at least 87.2% identity to SEQ ID NO:2;
(b) a polypeptide having at least one and not more than four repeat units of the reflectin polypeptide;
(c) a polypeptide comprising at least six repeat units of the reflectin polypeptide; and
(d) any combination of (a) through (c) combining the first component with at least a second component to form a biomimetic reflective material.

5. The method of claim 4, wherein the second component comprises a member of the group consisting of: a metal, an ion, a polymer, a fabric, a crystal, a fiber and a plastic.

6. A method of producing a biomimetic reflective material comprising causing expression in an isolated cell, of at least one polypeptide selected from the group consisting of:
(a) a reflectin polypeptide having at least 87.2% identity to SEQ ID NO:2;
(b) a polypeptide having at least one and not more than four repeat units of the reflectin polypeptide;
(c) a polypeptide comprising at least six repeat units of the reflectin polypeptide; and
(d) any combination of (a) through (c) using the isolated cell or extract thereof in producing a biomimetic reflective material.

7. The method of claim 6, wherein the isolated cell is selected from the group consisting of: a plant cell, a bacterial cell; a fungal cell, and an animal cell.

8. The biomimetic reflective material of claim 1, wherein the reflectin polypeptide has a predicted isoelectric point above 8.0.

9. The biomimetic reflective material of claim 1, wherein said polypeptide has an activity of a reflectin protein.