Rare earth element binding protein

Abstract

This invention relates to rare earth element binding protein and methods of recovering a rare earth element (REE) from a sample. The (REE) binding protein comprises the repeating sequence X1X2X3X4X5X6X7X8X9 wherein X denotes any amino acid, and X1 is D or E, X2 is A, T, or S, X3 is D, E or N, X4 is G, A, or F, X5 is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N.

Description

RARE EARTH ELEMENT BINDING PROTEIN Cross-Reference to Related Applications

[0001] The present application claims the benefit of the filing date of U.S. Provisional Application Serial No. 63 / 656, 161, filed June 5, 2024, the entire teachings of which application is hereby incorporated herein by reference. Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under contract number FA8650-22-C- 7213 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention. Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 5, 2025, is named BAT271PCT_SL.xml and is 9,323 bytes in size. Field

[0004] This invention relates to rare earth element binding protein and methods of recovering a rare earth element (REE) from a sample. Background

[0005] A challenge in establishing a more diversified REE supply chain is the difficulty of achieving cost-effective and environmentally sustainable REE extraction and separation from ore deposits and REE-containing waste. The current industrial REE production processes generate radioactive wastes, high volumes of acidic effluents, and organic solvents, resulting in a severeenvironmental burden. To alleviate supply vulnerability and diversify the global REE production chain, new processing technologies, specifically based in biological advancements enabling green REE extraction from alternative REE resources, are desired.

[0006] Proteins offer highly specific environments for metal-biological interactions to occur, so there is significant interest in their use for eco-friendly REE separation and purification. One such protein of interest is Lanmodulin (LanM), which reportedly demonstrates significantly higher binding affinity for REEs compared to calcium and other contaminant metals. However, LanM only binds 1-2 REEs when immobilized and exhibits relatively poor selectivity for individual REEs. See, e.g., WO2022 / 266120 entitled Compositions Comprising Proteins And Methods OF Use Thereof For Rare Earth Element Separation. Summary

[0007] A rare earth element (REE) binding protein comprising the repeating sequence X1X2X3X4X5X6X7X8X9wherein X denotes any amino acid, and X1is D or E, X2is A, T, or S, X3is D, E or N, X4 is G, A, or F, X5 is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N.

[0008] A rare earth element (REE) binding protein wherein the REE-binding protein comprises a sequence with at least 75% identity to SEQ ID NO. 1.

[0009] A method of recovering a rare earth element (REE) from a sample comprising: a. introducing a REE-binding protein to the sample wherein the REE-binding protein comprises the repeating sequence X1X2X3X4X5X6X7X8X9 wherein X denotes any amino acid, and X1is D or E, X2is A, T, or S, X3is D, E or N, X4is G, A, or F, X5is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N;b. recovering the REE from the sample.

[0010] A method of recovering a rare earth element (REE) from a sample comprising: a. introducing a REE-binding protein to the sample wherein the REE-binding protein comprises a sequence with at least 75% identity to SEQ ID NO. 1; and b. recovering the REE from the sample.

[0011] A method of recovering a rare earth element (REE) from a sample comprising: a. providing REE-binding protein immobilized on a solid matrix where the REE binding protein comprises the repeating sequence X1X2X3X4X5X6X7X8X9wherein X denotes any amino acid, and X1 is D or E, X2 is A, T, or S, X3 is D, E or N, X4 is G, A, or F, X5 is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9is D, E, or N; b. introducing a sample containing one or more REEs onto said REE-binding protein immobilized on said solid matrix; c. loading said one or more REEs onto said REE-binding protein immobilized on said solid matrix; d. unloading said one or more REEs from said REE-binding protein immobilized on said solid matrix.

[0012] A method of recovering a rare earth element (REE) from a sample comprising: a. providing REE-binding protein immobilized on a solid matrix where the REE- binding protein comprises a sequence with at least 75% identity to SEQ ID NO. 1 b. introducing a sample containing one or more REEs onto said REE-binding protein immobilized on said solid matrix;c. loading said one or more REEs onto said REE-binding protein immobilized on said solid matrix; and d. unloading said one or more REEs from said REE-binding protein immobilized on said solid matrix. Drawings

[0013] FIG. 1 provides an AlaphaFold2 model of the REE-binding protein comprising SEQ ID NO. 1 (HEW5) .

[0014] FIG.2 illustrates the binding performance of the REE-binding protein comprising SEQ ID NO. 1 (HEW5).

[0015] FIG.3 illustrates the separation of the indicated REEs using an immobilized REE-binding protein having SEQ ID NO. 1.

[0016] FIG. 4 depicts separation of pre-filtered Ce-removed simulated leachate using immobilized SEQ ID NO. 1 via the Halotag system (SEQ ID NO. 4) using a pH gradient. The figure shows ICP-MS validated data for a single cycle. Each data point was obtained via ICP-MS analysis.

[0017] FIG. 5 illustrates recyclability of SEQ ID NO. 1 by binding and eluting Nd.

[0018] FIG. 6 describes binding capacity of SEQ ID NO. 1 column determined via saturation to be nearly 17 µmoles of REE binding capacity. The shaded area represents the eluted fractions used in the calculation.

[0019] FIG 7a demonstrates that the individual repeating domains (i.e. X1-X9) are functional albeit have less REE loading capacity.

[0020] FIG. 7b shows that the selectivity of the individual domains does not change much compared to full length HEW5.Detailed Description Of Preferred Embodiments

[0021] REEs comprise a group of metals including lanthanides, yttrium (Y), and scandium (Sc). The lanthanides (or lanthanoids) are elements with atomic numbers 57 through 71 (e.g., lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium(Tb), dysprosium (Dy), holmium (Flo), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu), respectively).

[0022] The present invention provides a REE-binding protein, which may be utilized for example, in the recovery of REEs from a sample. The preferred REE herein has a molecular weight of 11.7 kDa and has eight (8) metal binding sites. The protein preferably binds REEs in the range of 10 µM to 50 µM. In addition, the REE-binding protein herein preferably provides a selectivity toward light rare earth elements (i.e., La, Ce, Pr, Nd, Pm, Sm, Eu, Gd) as compared to heavy rare earth elements (i.e., Tb, Dy, Ho, Er, Tm, Yb, Lu). More preferably, the preference amounts to a 2-4 fold selectivity preference toward light REE compared to heavy REE. The REE-binding protein herein also preferably allows for REE separation without the use of chelators.

[0023] The REE binding protein herein (HEW5) may first be described as continuous sequence of at least nine (9) amino acids with the sequence X1X2X3X4X5X6X7X8X9wherein X denotes any amino acid, and X1 is D or E, X2 is A, T, or S, X3 is D, E or N, X4 is G, A, or F, X5 is D, X6 is G, S, or D, X7is Y, L, V, F, I, E or W, X8is A, V, I, L, F or T, X9is D, E, or N. More preferably, the aforementioned sequence is contemplated to repeat 2, 4, 6, 8, 10 or 12 times, and such repeating sequence is preferably separated by at least four (4) amino acids. HEW5 contains 8 REE binding sites – the capacity was empirically determined to be 17 µmol by overloading the HEW5 column with La, washing unbound REE and then eluting at pH 3.0. Notably, this capacity matches thetheoretical molar capacity based on the 8 binding sites and amount of HEW5 loaded per mL beads in the column.

[0024] We have also demonstrated that the individual domain within HEW5 (i.e. X1-X9) is functional. Halo-HEW3.2 contains two metal binding sites, Halo-HEW1.6 contains a single metal binding site. Both can be immobilized and are capable of binding REEs, though the total amount of REEs decreases with decreasing number of binding sites. However, the proteins still show selectivity similar to full length HEW5, with slight differences. Additionally, a short peptide was synthesized that contains the same X1-X9motif, immobilized via an incorporated lysine residue, and characterized. It showed similar binding capacity as Halo-HEW1.6.

[0025] The REE-binding protein herein is preferably truncated from the 137 amino acid full-length protein from Nocardioides zeae (SEQ ID NO. 2). The REE-binding protein therefore preferably has the domain sequence selected from SEQ ID NO.1 and can be expressed in E. coli using coding SEQ ID NO. 3.: SEQ ID NO 1 (TRUNCATED HEW5) LENGTH: 112 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Pro Ser Ser Thr Glu Tyr Asp Ala Asp Gly Asp Gly Tyr Val Asp 1 5 10 15 Thr Arg Glu Ser Asp Thr Asp Gly Asp Gly Tyr Val Asp Thr Ile 20 25 30 Glu Thr Asp Thr Asp Gly Asp Gly Trp Val Asp Thr Val Ala Thr 35 40 45 Asp Thr Asp Gly Asp Gly Tyr Ile Asp Thr Val Ala Thr Asp Thr 50 55 60 Asp Gly Asp Gly Tyr Ala Asp Val Val Glu Thr Asp Thr Asp Gly 65 70 75 Asp Gly Tyr Thr Asp Glu Val Ala Tyr Asp Ala Asp Gly Asp Gly 80 85 90 Tyr Ile Asp Thr Val Glu Ala Asp Thr Asp Gly Asp Gly Tyr Thr 95 100 105 Asp Thr Val Val His Asp Gly 110SEQ ID NO 2 (WT HEW5) LENGTH: 137 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Tyr Ala Ser Asn Ala Glu Pro Thr Pro Pro Pro Ala Pro Ser 1 5 10 15 Ser Thr Glu Tyr Asp Ala Asp Gly Asp Gly Tyr Val Asp Thr Arg 20 25 30 Glu Ser Asp Thr Asp Gly Asp Gly Tyr Val Asp Thr Ile Glu Thr 35 40 45 Asp Thr Asp Gly Asp Gly Trp Val Asp Thr Val Ala Thr Asp Thr 50 55 60 Asp Gly Asp Gly Tyr Ile Asp Thr Val Ala Thr Asp Thr Asp Gly 65 70 75 Asp Gly Tyr Ala Asp Val Val Glu Thr Asp Thr Asp Gly Asp Gly 80 85 90 Tyr Thr Asp Glu Val Ala Tyr Asp Ala Asp Gly Asp Gly Tyr Ile 95 100 105 Asp Thr Val Glu Ala Asp Thr Asp Gly Asp Gly Tyr Thr Asp Thr 110 115 120 Val Val His Asp Gly Ala Ser Asp Ser Gly Leu Glu Ser Thr Leu 125 130 135 Asp Ala SEQ ID NO 3 (E. coli HEW5) LENGTH: 117 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Gly Ser Gly Pro Ser Ser Thr Glu Tyr Asp Ala Asp Gly Asp 1 5 10 15 Gly Tyr Val Asp Thr Arg Glu Ser Asp Thr Asp Gly Asp Gly Tyr 20 25 30 Val Asp Thr Ile Glu Thr Asp Thr Asp Gly Asp Gly Trp Val Asp 35 40 45 Thr Val Ala Thr Asp Thr Asp Gly Asp Gly Tyr Ile Asp Thr Val 50 55 60 Ala Thr Asp Thr Asp Gly Asp Gly Tyr Ala Asp Val Val Glu Thr 65 70 75 Asp Thr Asp Gly Asp Gly Tyr Thr Asp Glu Val Ala Tyr Asp Ala 80 85 90 Asp Gly Asp Gly Tyr Ile Asp Thr Val Glu Ala Asp Thr Asp Gly 95 100 105 Asp Gly Tyr Thr Asp Thr Val Val His Asp Gly Ser 110 115SEQ ID NO 4 (HALO HEW5) LENGTH: 420 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr 1 5 10 15 Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro 20 25 30 Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser 35 40 45 Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro Thr His 50 55 60 Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys 65 70 75 Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp 80 85 90 Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile 95 100 105 His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn 110 115 120 Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro 125 130 135 Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe 140 145 150 Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp 155 160 165 Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg 170 175 180 Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu 185 190 195 Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu 200 205 210 Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu 215 220 225 Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe 230 235 240 Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg 245 250 255 Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro 260 265 270 Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser 275 280 285 Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Gly Ser 290 295 300 Gly Gly Ser Gly Ser Gly Ser Gly Pro Ser Ser Thr Glu Tyr Asp 305 310 315 Ala Asp Gly Asp Gly Tyr Val Asp Thr Arg Glu Ser Asp Thr Asp 320 325 330 Gly Asp Gly Tyr Val Asp Thr Ile Glu Thr Asp Thr Asp Gly Asp 335 340 345 Gly Trp Val Asp Thr Val Ala Thr Asp Thr Asp Gly Asp Gly Tyr 350 355 360Ile Asp Thr Val Ala Thr Asp Thr Asp Gly Asp Gly Tyr Ala Asp 365 370 375 Val Val Glu Thr Asp Thr Asp Gly Asp Gly Tyr Thr Asp Glu Val 380 385 390 Ala Tyr Asp Ala Asp Gly Asp Gly Tyr Ile Asp Thr Val Glu Ala 395 400 405 Asp Thr Asp Gly Asp Gly Tyr Thr Asp Thr Val Val His Asp Gly 410 415 420 SEQ ID NO 5 (CBD HEW5) LENGTH: 176 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Ser Ser Gly Ser Thr Asn Pro Gly Val Ser Ala Trp Gln Val 1 5 10 15 Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys 20 25 30 Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp Glu 35 40 45 Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln Gly Ser Ser Gly 50 55 60 Ser Ser Ser Gly Pro Ser Ser Thr Glu Tyr Asp Ala Asp Gly Asp 65 70 75 Gly Tyr Val Asp Thr Arg Glu Ser Asp Thr Asp Gly Asp Gly Tyr 80 85 90 Val Asp Thr Ile Glu Thr Asp Thr Asp Gly Asp Gly Trp Val Asp 95 100 105 Thr Val Ala Thr Asp Thr Asp Gly Asp Gly Tyr Ile Asp Thr Val 110 115 120 Ala Thr Asp Thr Asp Gly Asp Gly Tyr Ala Asp Val Val Glu Thr 125 130 135 Asp Thr Asp Gly Asp Gly Tyr Thr Asp Glu Val Ala Tyr Asp Ala 140 145 150 Asp Gly Asp Gly Tyr Ile Asp Thr Val Glu Ala Asp Thr Asp Gly 155 160 165 Asp Gly Tyr Thr Asp Thr Val Val His Asp Gly 170 175 SEQ ID NO 6 (HALO HEW3.2) LENGTH: 342 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr 1 5 10 15 Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro 20 25 30 Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser 35 40 45 Ser Tyr Val TrpHis 50 55 60Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys 65 70 75 Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp 80 85 90 Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile 95 100 105 His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn 110 115 120 Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro 125 130 135 Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe 140 145 150 Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp 155 160 165 Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg 170 175 180 Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu 185 190 195 Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu 200 205 210 Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu 215 220 225 Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe 230 235 240 Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg 245 250 255 Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro 260 265 270 Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser 275 280 285 Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Gly Ser 290 295 300 Gly Gly Ser Gly Ser Gly Ser Gly Pro Ser Ser Thr Glu Tyr Asp 305 310 315 Ala Asp Gly Asp Gly Tyr Val Asp Thr Arg Glu Ser Asp Thr Asp 320 325 330 Gly Asp Gly Tyr Val Asp Thr Ile Glu Thr Asp Thr 335 340 SEQ ID NO 7(HALO HEW1.6) LENGTH: 329 TYPE: PRT ORGANISM: Nocardioides zeae SEQUENCE: 1 Met Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr 1 5 10 15 Val Glu Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro 20 25 30 Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser 35 40 45 Ser Tyr Val TrpHis 50 60Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp Lys 65 70 75 Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp 80 85 90 Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile 95 100 105 His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn 110 115 120 Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro 125 130 135 Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe 140 145 150 Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp 155 160 165 Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg 170 175 180 Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu 185 190 195 Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu 200 205 210 Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu 215 220 225 Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe 230 235 240 Trp Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg 245 250 255 Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro 260 265 270 Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser 275 280 285 Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly Gly Ser 290 295 300 Gly Gly Ser Gly Ser Gly Ser Gly Pro Ser Ser Thr Glu Tyr Asp 305 310 315 Ala Asp Gly Asp Gly Tyr Val Asp Thr Arg Glu Ser Asp Thr 320 325 SEQ ID NO 8(HALO HEW1.6 Peptide) LENGTH: 25 TYPE: PRT ORGANISM: NA SEQUENCE: 1 Ser Lys Ser Gly Pro Ser Ser Thr Glu Tyr Asp Ala Asp Gly Asp Gly Tyr Val Asp 1 5 10 15 Thr Arg Glu Ser Asp Thr 20 25

[0026] In certain embodiments, the REE-binding protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO.1, or a sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO.1.

[0027] FIG.1 provides an AlphaFold2 model of the REE-binding protein herein comprising SEQ ID NO.1. FIG 2 illustrates the binding performance of the REE-binding protein comprising SEQ ID. NO. 1 illustrating the selectivity towards light REEs as compared to heavy REEs.

[0028] Immobilization of the REE-binding protein herein on a solid matrix was confirmed to facilitate REE separation in a continuous flow process. A tagged REE-binding protein having SEQ ID. NO. 1 can be immobilized on a solid matrix such as a packed bead matrix via a non-covalent linkage (Chitin-binding domain via SEQ ID NO. 5 interaction with chitin) or a covalent linkage (performed through Halotag-based immobilization via SEQ ID NO. 4 or directly via lysine in the chitin binding domain of SEQ ID NO.5 to a bead bearing an oxirane functional group) and a buffer (e.g., pH 5.5) is continuously flowed through the column. Over time, REEs are released from the column using a lower pH buffer (e.g., pH 3.0).

[0029] FIG.3 illustrates the separation of the indicated REEs using the immobilized REE-binding protein having SEQ ID. NO. 1 in a continuous flow system. As can be observed, in a single run three (3) different light REEs could be partially separated, with each nearing 50% recovery and a significant increase in purity compared to the leachate.

[0030] FIG. 4 illustrates the separation of La from Pr and Nd using a mock industrial feedstock, reaching >98% purity of La in a single column run, which demonstrates SEQ ID NO.1’s industrial utility to separate REEs in commercial feedstocks. Importantly, all impurities (Fe, Sr, Ca, and Mg) were removed early in the pH 5.5 wash. Lastly, stability of the REE-binding protein is important for industrial applications since recovery and reuse of the immobilized protein would reduce process costs.

[0031] FIG. 5 demonstrates that SEQ ID. No. 1 is capable of > 10 bind and release cycles of Nd without loss in binding efficiency, indicating that this protein is relatively stable and can be reused for multiple cycles.

[0032] FIG.6 describes binding capacity of HEW5 column determined via saturation to be nearly 17 µmoles of REE binding capacity. The shaded area represents the eluted fractions used in the calculation.

[0033] FIG. 7a shows the binding capacity comparison of Halo-HEW5 (8 binding sites), Halo- HEW3.2 (2 binding sites) and a HEW1.6 (1 binding site) in the form of a recombinant fusion (Halo-HEW1.6) and solid-phase synthesized peptide (HEW1.6 peptide). FIG. 7b demonstrates that the truncated proteins display selectivity for different REEs with similar trends as HEW5, albeit some relatively slight differences were observed.

Claims

Claims:

1. A rare earth element (REE) binding protein comprising the repeating sequence X1X2X3X4X5X6X7X8X9wherein X denotes any amino acid, and X1is D or E, X2is A, T, or S, X3 is D, E or N, X4 is G, A, or F, X5 is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N.

2. A rare earth element (REE) binding protein wherein the REE binding protein comprises a sequence with at least 75% identity to SEQ ID. NO.

1.

3. The REE binding protein of claim 1 or 2 wherein the REE binding protein is immobilized on a solid matrix.

4. The REE binding protein of claim 1 wherein said sequence repeats 2, 4, 6, 8, 10 or 12 times.

5. The REE binding protecting of claim 1 wherein the sequence is separated by at least four (4) amino acids.

6. The REE binding protein of claim 1 or 2 wherein the REE binding protein is immobilized on said solid matrix via a non-covalent linkage.

7. The REE binding protein of claim 1 or 2 wherein the REE binding protein is immobilized on said solid matrix via a covalent linkage.

8. A method of recovering a rare earth element (REE) from a sample comprising: a. introducing a REE-binding protein to the sample wherein the REE-binding protein comprises a repeating sequence X1X2X3X4X5X6X7X8X9wherein X denotes any amino acid, and X1is D or E, X2is A, T, or S, X3is D, E or N, X4is G, A, or F, X5is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N;b. recovering the REE from the sample.

9. The method of claim 7 further comprising purifying a REE from the sample.

10. The method of claim 7 wherein the REE-binding protein is produced in a host cell and truncated.

11. The method of claim 7 wherein the REE-binding protein indicates a difference in selectivity toward light REEs versus heavy REEs.

12. The method of claim 7 wherein said repeating sequence repeats 2, 4, 6, 8, 10 or 12 times.

13. The method of claim 7 wherein the repeating sequence is separated by at least four (4) amino acids.

14. The method of claim 10 wherein the light REE comprises La, Ce, Pr, Nd, Pm, Sm, Eu, or Gd.

15. The method of claim 10 wherein the REE comprises a heavy rare earth element comprising Tb, Dy, Ho, Er, Tm, Yb, or Lu.

16. A method of recovering a rare earth element (REE) from a sample comprising: a. introducing a REE-binding protein to the sample wherein the REE-binding protein comprises a sequence with at least 75% identity to SEQ ID NO. 1; and b. recovering the REE from the sample.

17. The method of claim 16 wherein the REE-binding protein indicates a difference in selectivity toward light REEs versus heavy REEs.

18. The method of claim 17 wherein the light REE comprises La, Ce, Pr, Nd, Pm, Sm, Eu, or Gd.

19. The method of claim 17 wherein the heavy REE comprises Tb, Dy, Ho, Er, Tm, Yb, or Lu.

20. A method of recovering a rare earth element (REE) from a sample comprising:a. providing REE-binding protein immobilized on a solid matrix where the REE binding protein comprises the repeating sequence X1X2X3X4X5X6X7X8X9 wherein X denotes any amino acid, and X1is D or E, X2is A, T, or S, X3is D, E or N, X4is G, A, or F, X5 is D, X6 is G, S, or D, X7 is Y, L, V, F, I, E or W, X8 is A, V, I, L, F or T, X9 is D, E, or N; b. introducing a sample containing one or more REEs onto said REE-binding protein immobilized on said solid matrix; c. loading said one or more REEs onto said REE-binding protein immobilized on said solid matrix; d. unloading said one or more REEs from said REE-binding protein immobilized on said solid matrix.

21. The method of claim 20 wherein said unloading of said one or more REEs from said REE- binding protein comprises adjusting pH.

22. A method of recovering a rare earth element (REE) from a sample comprising: a. providing REE-binding protein immobilized on a solid matrix where the REE- binding protein comprises a sequence with at least 75% identity to SEQ ID NO. 1 b. introducing a sample containing one or more REEs onto said REE-binding protein immobilized on said solid matrix; c. loading said one or more REEs onto said REE-binding protein immobilized on said solid matrix; and d. unloading said one or more REEs from said REE-binding protein immobilized on said solid matrix.

23. The method of claim 22 wherein said unloading of said one or more REEs from said REE- binding protein comprises adjusting pH.

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