Purification of peptides and proteins using affinity tags

Abstract

Described are affinity tags comprising at least one non-canonical amino acid residue in D-enantiomer form. The affinity tags can comprise at least one histidine in D-form. The affinity tags demonstrate metal affinity substantially similar to that of their corresponding L forms. The affinity tags with at least one histidine in D-form have reduced recognition by antibodies recognizing the L-forms of the tags. Also described are methods for purifying peptides and proteins using the affinity tags. The affinity tags are improved affinity tags for protein purification.

Description

Attorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1PURIFICATION OF PEPTIDES AND PROTEINS USING AFFINITY TAGSREEATED APPEICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 722,301, filed November 19, 2024, the contents of which are incorporated herein by reference in their entirety.TECHNICAE FIEED

[0002] Disclosed herein are peptide and protein affinity purification tags and methods for purification of peptides and proteins containing one or more non-canonical amino acids for biotechnological and therapeutic applications.BACKGROUND

[0003] A widely used protein purification method of immobilized metal- affinity chromatography (IMAC) produces highly purified proteins. In some instances, the proteins obtained via IMAC have been purified to over 90% purity with only one cycle of purification. The purification using IMAC is based on the interactions between a transition metal ion, such as Co2+, Ni2+, Cu2+, Zn2+, immobilized on a matrix, and specific amino acid side chains. Histidine exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal.

[0004] IMAC purification method does not perform well when the peptides or proteins tagged for purification are rare proteins or are proteins in highly heterogenous medium. In these cases, affinity purification with IMAC, coupled with other protein purification methods, is used. There remains a need for new protein purification methods and tags for biotechnological and therapeutic use. The present disclosure addresses these needs.SUMMARY

[0005] In certain aspects, the present disclosure provides affinity tags for protein purification comprising at least one amino acid in D-enantiomer form.

[0006] In certain aspects, the present disclosure provides affinity tags for protein purification comprising an amino acid sequence having about 70% to about 100% identity toAttomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 any one of the amino acid sequences set forth in SEQ ID NO: 1 or 2 with at least one amino acid residue in D-enantiomer form.

[0007] In certain aspects, the present disclosure provides synthetic peptides comprising an affinity tag of the present disclosure.

[0008] In certain aspects, the present disclosure provides methods of producing a tagged peptide or protein, comprising linking a peptide or protein to an affinity tag comprising at least one amino acid in D-enantiomeric form, thereby producing the tagged peptide or protein.

[0009] In certain aspects, the present disclosure provides methods of purifying a peptide or protein comprising an affinity tag comprising at least one amino acid in D- enantiomeric form, the methods comprising the steps of: a. equilibrating with equilibration buffer a resin having any one of metal ions Co2+, Ni2+, Cu2+, or Zn2+immobilized in the resin; b. loading the resin with a solution containing the peptide or protein to bind the peptide or protein to the resin; c. washing the resin with wash buffer; and d. eluting bound peptide or protein by passing elution buffer through the resin and collecting the eluate comprising the purified peptide or protein.

[0010] In certain aspects, the present disclosure provides methods comprising using the affinity tag of the present disclosure; wherein the method is selected from the group consisting of protein tagging, protein purification, a diagnostic method, and a therapeutic method.

[0011] The present disclosure provides affinity purification tags comprising at least one amino acid in D-enantiomer form for purifying peptides and proteins.

[0012] Also disclosed are synthetic peptides and proteins comprising between about 0.5% and about 100% of amino acid residues in D-enantiomer form and the affinity purification tags. The affinity purification tag can be linked to the synthetic peptides via a non-cleavable linker.

[0013] Also disclosed are methods of making and using the affinity purification tags and methods of purifying peptides and proteins in D-enantiomeric form using affinity tags.

[0014] Other aspects and embodiments of the invention will be readily apparent from the following detailed description of the invention.BRIEF DESCRIPTION OF THE DRAWINGSAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1

[0015] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed compositions and methods there are shown in the drawings exemplary embodiments of compositions and methods; however, these should not be limited to the specific embodiments disclosed. In the drawings:

[0016] Figure 1 is a graph depicting absorbance (450) over antibody concentration (M) of the absorbance data from the assayed His-tags (L, L-scramble, D-retro-inverted, D- retro-inverted-scramble, as listed in Table 1). Strong antibody binding is observed to the L- Hise-tag, whereas binding of the same antibody is very low or undetected to the D-Hise-tag. Scrambled versions of the tag containing intermittent insertions of Gly or Ser between the His residues are also not recognized, either in their L- enantiomer form (L-form) or D-enantiomer form (D-form).

[0017] Figure 2 is a graph depicting absorbance (AU450) over concentration (M) of [Ni2+-HRP] obtained from Ni2+-NTA-HRP ELISA kits. The data show equipotent binding of both biotinylated L-Hise and biotinylated D-Hise peptides listed in Table 1.

[0018] Figure 3A shows TIC and UV chromatograms for crude extract (top two panels, respectively) and for NiNTA purified (lower two panels, respectively) for purification of D-6xHis-RFX-Vl peptide (“D-RFX-V1”).

[0019] Figure 3B shows the mass spectrum and ion envelope for the NiNTA purified D-6xHis-RFX-Vl.

[0020] Figure 4 is a representative diagram depicting a general synthetic scheme for linking a peptide to an affinity tag or to another peptide through a non-cleavable linker. Also depicted is an exemplary workflow of tagged protein purification. The purification steps of recombinantly generated tagged peptides or chemically synthesized (via SPPS) tagged peptides are shown.

[0021] Figure 5 depicts various exemplary peptides linked to an affinity tag.Attomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1DETAILED DESCRIPTION

[0022] The disclosed compositions and methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed compositions and methods are not limited to the specific compositions and methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed compositions and methods.

[0023] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed compositions and methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0024] Throughout this text, the descriptions refer to compositions and methods of using said compositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using said composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

[0025] It is to be appreciated that certain features of the disclosed compositions and methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0026] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to thoseAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 described herein can be used in practice or testing. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0028] As used herein, the term “substantial” or “substantially” refers to a degree of similarity, difference, increase, or decrease, as in a comparison to a known value. Substantial can include at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% similarity, difference, increase, or decrease, as in a comparison to a known value.

[0029] It is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, or ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0030] As used herein, approximating language may be applied to modify any quantitative representation that may vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by “about” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0031] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than theAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0032] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints (e.g., “between 2 grams and 10 grams, and all the intermediate values includes 2 grams, 10 grams, and all intermediate values”). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values. All ranges are combinable.

[0033] Further, the term “comprising” should be understood as having its open- ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B may be a composition that includes A, B, and other components, but may also be a composition made of A and B only.

[0034] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0035] ‘ ‘Identical” or “identity” as used herein in the context of two peptide or two polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0036] As used herein “D-enantiomer form” in the context of amino acids refers to stereoisomeric forms of L-amino acids. D-enantiomers of amino acids (also called D-amino acids) can be referred to by the same letter as their corresponding L-enantiomer (also called L-amino acid), but in lower case. Thus, for example, the L-enantiomer of arginine is referredAttorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 to as “R”, while the D-enantiomer can be referred to as “r.” Unless specifically defined, all amino acids and residues are in L-enantiomer form.

[0037] As used herein, the term “flanked by”, when used in reference to a given amino acid residue, refers to the identity of the amino acid residues on either side of the given amino acid residue. For example, a histidine flanked by glycine refers to the amino acid sequence Gly-His-Gly; and a proline flanked by lysine refers to the amino acid sequence Lys- Pro-Lys. A given amino acid residue may also be flanked by nonidentical amino acid residues. For example, a proline flanked by lysine and histidine may refer to the amino acid sequences N-Lys-Pro-His-C or N-His-Pro-Lys-C; wherein N and C refer to the directions of the N and C termini with respect to the peptide sequence. The amino acid residues on either side of the given amino acid residue may alternatively be said to be “flanking” the given amino acid residue.Affinity Tags in D-Enantiomer Form

[0038] Disclosed are affinity tags for protein purification. The affinity tags can comprise at least one non-canonical amino acid. The non-canonical amino acids may be D- enantiomer forms of naturally occurring L-amino acids. The non-canonical amino acids may be chemically modified natural amino acids. For example, the non-canonical amino acids include, but are not limited to chemically modified tyrosine, leucine, alanine, methionine, phenylalanine, lysine, arginine, glutamine, serine, glycine, and histidine. For example, the non-canonical amino acids include, but are not limited to thiazolylalanine (Taz), such as 4- (thiazolyl)-L-alanine (Thz).The non-canonical amino acids can be D-enantiomer forms of alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, or glutamine. In some aspects, the non-canonical amino acids can be D- enantiomer forms of tyrosine, leucine, alanine, methionine, phenylalanine, lysine, arginine, glutamine, serine, and histidine. In some aspects, the non-canonical amino acids can be D- enantiomer forms of tryptophan, histidine, cysteine, and arginine. The affinity tags can comprise tryptophan in D-form. The affinity tags can comprise histidine in D-form. The affinity tags can comprise cysteine in D-form. The affinity tags can comprise arginine in D- form. The affinity tags can comprise one or more of tryptophan, histidine, cysteine, and arginine in D-form.

[0039] The affinity tags can comprise at least one amino acid in D-enantiomer form. In some embodiments, the at least one amino acid in D-enantiomer form is a histidine residueAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 in D-enantiomer form. The affinity tag comprising at least one amino acid in D-enantiomer form can have substantially no binding to antibodies raised against an affinity tag having the same amino acids and having all amino acid residues in L-enantiomer form.

[0040] In some embodiments, the affinity tags comprise one or more zinc finger domains. The zinc finger domains are short peptide repeats having a conserved pair of cysteines and histidines at defined positions. In some embodiments, the affinity tags can comprise an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, or 22. For example, the affinity tags can comprise one or more of C2H2 (Cys2His2) (SEQ ID NO: 16) zinc finger domain, Cys3His (SEQ ID NO: 17) domain, Cys4 (SEQ ID NO: 18) domain, Cys6 (SEQ ID NO: 19) domain, Cys-X2-Cys-X4-His-X4-Cys (SEQ ID NO: 20) domain, CCHC (SEQ ID NO: 21) domain, or CPXCG (SEQ ID NO: 22) domain, where X is any canonical or non- canonical amino acid. The affinity tags comprising zinc finger domains can comprise one or more amino acids in D-enantiomer form. For example, any one, two, three, four, or more amino acids in the zinc finger domain can comprise the respective amino acid in D-form.

[0041] In some embodiments, the affinity tag comprises an amino acid sequence having about 70% to about 100% identity, about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 3, 4, or 6.

[0042] In some embodiments, the affinity tag is modified glutathione synthetase (GST). The modified GST comprises one or more amino acids in D-form.

[0043] In some embodiments the affinity tag is modified Strep-tagll or modified twin Strep-tag. The modified Strep-tagll or modified twin Strep-tag comprises one or more amino acids in D-form.

[0044] In some embodiments, the affinity tags comprise histidine. In some embodiments, the affinity tags can comprise between one and 12 histidine residues in D- enantiomer form (D-histidine). The affinity tags can comprise between two and three histidine residues in D-enantiomer form. The histidine residues in D- or L- enantiomer form can be flanked by an amino acid residue that is not a histidine. In some aspects, the amino acid residue that is not a histidine is a serine residue in D- and / or L-enantiomer form. For example, the affinity tags can include up to 20 amino acid residues comprising one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or more D-histidines. These one or more histidine residues in the affinity tag can be flanked by a serine and / or a glycine amino acid residue, the serine residue in D- and / or L-enantiomer form. In some embodiments, theAttomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 affinity tags comprise an amino acid sequence having about 70% to about 100% identity to a sequence set forth in SEQ ID NO: 10 (hsghgshshghgsh).

[0045] The affinity tags can comprise homochiral segments of amino acids. The homochiral segments are segments with all the amino acid residues in the segment in L-form or all the amino acid residues in the segment in D-form. In some embodiments, the affinity tags can comprise homochiral segments of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12 amino acids. Examples of affinity tags comprising a homochiral segment are affinity tags having an amino acid sequence comprising about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 23 or 24. For example, the affinity tag may have an amino acid sequence comprising at least five L-histidines (HHHHH, SEQ ID NO: 23), or at least five D-histidines (hhhhh, SEQ ID NO: 24).

[0046] In some embodiments, the affinity tags comprise an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 3 and 4 with substantially all of the amino acid residues in D-enantiomer form, as shown in Table 1. Additional exemplary affinity tags with homochiral segments comprise amino acid sequences having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 or 3, as shown in Table 1.

[0047] In some embodiments, the affinity tags have between 2 and 10 histidine residues in D-enantiomeric form. In some embodiments, the affinity tags have between 2 and 6 histidine residues in D-enantiomeric form.

[0048] The disclosed affinity tags can comprise between one and 12 histidine residues in D-enantiomer form and a dissociation constant (KD) from Ni2+ions of between about 0.1 nM and about 1 pM. For example, the KD from Ni2+ions can be between about 0.1 nM and about 1 pM, between about 0.5 nM and about 1 pM, between about 1 nM and about 1 pM, between about 1 nM and about 500 nM, between about 1 nM and about 100 nM, between about 1 nM and about 5 nM, between about 1 nM and about 4 nM, or between about 1 nM and about 3 nM. In some embodiments, the KD from Ni2+ions is about 1 nM, about 2 nM, about 3 nM, or about 2.7 nM. In some embodiments, the KD from Ni2+ions is about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0,4, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM,Attomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, or about 1000 nM. The KD value can be measured using readily available in vitro methods, such as methods utilizing histidine detector compounds, as described in Example 2.

[0049] In some embodiments, the affinity tags comprising between one and 12 D- histidines have substantially no binding to antibodies raised against an affinity tag comprising six consecutive histidine residues in L-enantiomer form. For example, substantially no binding of the affinity tag comprising between two and 12 D-histidines to antibodies raised against an affinity tag comprising six consecutive histidine residues in L-enantiomer form is a binding with a KD of greater than about 1 nM, greater than about 1 pM, greater than about 10 pM, greater than about 100 pM, greater than about 1 mM, or greater than about 10 mM.

[0050] In other aspects, the disclosed affinity tags, peptides, or proteins may be tagged with other affinity tags. These include biotin, desthiobiotin, azidohomoalanine, and homopropargylglycine.Linkers

[0051] Also disclosed are non-cleavable linkers. In some aspects, the linkers are positioned at the N-terminal of a peptide. In some aspects, the linkers are positioned at the C- terminal of a peptide. The linkers can be reacted with an affinity tag to ligate a peptide to the affinity tag. The linker can be reacted with two peptides to ligate one peptide or protein to another peptide.

[0052] In some aspects, the linker is a non-cleavable linker. The non-cleavable linker is one that is generally resistant to dissociation from the peptide via chemical or enzymatic means. The non-cleavable linkers include amino acids, polyamino acids, polyethylene glycol (PEG) linkers such as bifunctional and heterobifunctional PEG linkers, and amino acid-functional PEG linkers. Examples of polyamino acid linkers include polyamino units repeated n times. In some aspects, polyamino acid linkers comprise amino acid sequences having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 11, 12, or 13. In some aspects, polyamino acid linkers include (GGGGS)n(SEQ ID NO: 11), (GSGS)n(SEQ ID NO: 12), and (HGSHGS)n(SEQ ID NO: 13). In some aspects, the n is a value between about 1 and about 20, such as about 1,Attorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.Methods of Making the Affinity Tags, Linkers, and Tagged Peptides and Proteins

[0053] Methods of making the disclosed affinity tags, peptides, and proteins include protein synthesis methods of solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL). The disclosed affinity tags, peptides, and proteins can first be generated as oligopeptide fragments via SPPS and then ligated via NCL.

[0054] An exemplary scheme of synthesizing a peptide or a protein with an affinity tag attached to the peptide or protein via a linker is shown in Figures 4. The linkers may be non-cleavable linkers. Examples of non-cleavable linkers are provided above.Methods of Using the Affinity Tags

[0055] The disclosed affinity tags, peptides, and proteins can be used in biotechnology and therapeutics. Suitable methods of use include protein tagging, protein purification, diagnostic methods, prophylactic methods, and therapeutic methods.

[0056] In some embodiments, the affinity tag for protein purification comprises an amino acid sequence having about 70% to about 100% identity, about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 or 2.

[0057] In some embodiments of the methods disclosed herein, the affinity tag comprises an amino acid sequence having about 70% to about 100% identity, about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 or 2.

[0058] In one aspect, the disclosed affinity tags are used in producing tagged peptides or tagged proteins. The disclosed affinity tags can be incorporated at the N-terminals of recombinant or synthetic peptides and proteins to produce the tagged peptides or tagged proteins. The disclosed affinity tags can be incorporated at the C-terminals of recombinant or synthetic peptides and proteins to produce the tagged peptides or tagged proteins. The tagged peptides or tagged proteins can be recombinantly or synthetically produced.

[0059] In some embodiments, the synthetic peptide comprises an amino acid sequence having about 70% to about 100% identity, about 80% to about 100% identity, aboutAttomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 14 or 15.

[0060] In some aspects, producing the tagged peptides or tagged proteins comprises linking the affinity tags to the peptides or proteins. The linking can comprise recombinantly producing the peptide or proteins with the affinity tag as part of one open reading frame. The linking can comprise chemically linking the affinity tag via a linker to a synthetic or recombinant peptide or to a synthetic or recombinant protein.

[0061] In some aspects, the linker comprises a non-cleavable linker. The non- cleavable linker can comprise amino acids, polyamino acids, bifunctional polyethylene glycol (PEG) linkers, heterobifunctional PEG linkers, or amino acid-functional PEG linkers.

[0062] In one aspect, the disclosed affinity tags are used in peptide or protein purification. The tagged recombinant peptides, tagged recombinant proteins, tagged synthetic peptides, and tagged synthetic proteins can be purified using the disclosed affinity tags.

[0063] In some aspects, the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins are in native form. In these aspects, the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins are maintained in native conditions with the use of one or more of neutral or pH-balanced buffers, optionally containing protease inhibitors, cofactor reagents, and reducing agents.

[0064] In some aspects, the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins are in denatured form. In these aspects, the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins are maintained under denaturing conditions in buffers containing reducing agent, protease inhibitors, denaturants, and detergents.

[0065] The tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins can then be purified with the use of affinity chromatography. In some aspects, the affinity chromatography is immobilized metalaffinity chromatography (IMAC). The metal ion can be a transition metal ion, such as cobalt (Co2+), nickel (Ni2+), copper (Cu2+), or zinc (Zn2+). In some aspects, the transition metal ion is immobilized on a substrate. Suitable substrates include resin column and resin beads, such as resin flow columns, resin spin column, or resin microbeads. In some aspects, the metal ions are coupled to nitrilotriacetic acid (NTA). Metal ion-NTA complex (such as Ni-NTA, Co- NTA, Cu-NTA, or Zn-NTA) can then be coupled to agarose or magnetic beads for IMAC.Attorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1

[0066] For example, the affinity-tagged peptides or proteins can be purified using the commercially available matrices nickel-nitrilotriacetic acid (Ni2+-NTA) and Co2+- carboxylmethylaspartate (Co2+-CMA), which are coupled to a solid support resin. These matrices securely coordinate metal ions through four coordination sites while leaving two of the transition metal coordination sites exposed to interact with histidine residues in the affinity tag. The stability of metal binding in these resins also allows the resins to be regenerated and reused several times. The Ni2+-NTA matrix (available from Qiagen®) has a binding capacity of 5-10 mg protein / ml of matrix resin and a high binding affinity for the six residue polyhistidine tag at pH 8.0. The Co2+-CMA matrix (Talon resin, available from Clontech®) exhibit less nonspecific protein binding than the Ni2+-NTA resin, resulting in higher elution product purity. The binding capacity of the Co2+-CMA resin is also about 5- 10 mg of protein / ml of resin (Bornhorst and Falk, Methods Enzymol. 2000 ; 326: 245-254).

[0067] The Zn-NTA can be used for purification peptides with affinity tags having zinc finger motifs.

[0068] Reduced glutathione (GSH), when immobilized as a ligand to agarose or other chromatography supports, enables high-yield and high-quality purification of recombinant proteins expressed as fusions with glutathione S-transferase (GST).

[0069] Modified Strep-Tactin, when immobilized as a ligand to agarose or other chromatography supports, enables high-yield and high-quality purification of recombinant proteins expressing modified Strep-tagll or modified twin Strep-tag.

[0070] The purification of the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins via IMAC can include the following steps.1. equilibrating with equilibration buffer a resin having any one of metal ions Co2+, Ni2+, Cu2+, or Zn2+immobilized in the resin;2. loading the resin with solution containing the tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins;3. washing the resin with wash buffer;4. eluting bound tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins by passing elution buffer through the resin to obtain an eluate;5. concentrating the eluate to obtain concentrated eluate; and6. dialyzing the concentrated eluate against storage buffer.Attomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1

[0071] In some aspects, the resin is in a spin column format. In these aspects, each one of the steps of equilibrating, loading, washing, and eluting are followed by centrifuging the spin columns.

[0072] In some aspects, the step of washing is repeated one or more times.

[0073] Equilibration buffer can be buffer suitable for storage and maintenance of tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins. In some aspects, equilibration buffer comprises neutral or pH-balanced buffer containing salts.

[0074] In some aspects, the equilibration buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the equilibration buffer can comprise 6 M urea. The equilibration buffer can comprise 8 M urea.

[0075] In some aspects, the equilibration buffer can be a buffer comprising guanidine hydrochloride (Gdn.HCl), such as between 4 M and 6 M Gdn.HCl. For example, the equilibration buffer can comprise 6 M Gdn.HCl. The wash buffer can be any buffer comprising up to about 50 mM imidazole. For example, wash buffer can contain between about 1 mM and about 50 mM imidazole, such as between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 mM, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM imidazole. Wash buffer can comprise about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM imidazole.

[0076] In some aspects, the wash buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the wash buffer can comprise 6 M urea. The wash buffer can comprise 8 M urea.

[0077] In some aspects, the wash buffer can be a buffer comprising Gdn.HCl, such as between 4 M and 6 M Gdn.HCl. For example, the wash buffer can comprise 6 M Gdn.HCl.

[0078] Elution buffer can be a buffer comprising up to about 500 mM imidazole, such as between about 200 mM and about 500 mM imidazole, such as about 250 mM imidazole.

[0079] In some aspects, the elution buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the elution buffer can comprise 6 M urea. The elution buffer can comprise 8 M urea.Attorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1

[0080] In some aspects, the elution buffer can be a buffer comprising Gdn.HCl, such as between 4 M and 6 M Gdn.HCl. For example, the elution buffer can comprise 6 M Gdn.HCl.

[0081] Storage buffer can be a buffer comprising neutral or pH-balanced buffer. Storage buffer can comprise salts and, optionally, protease inhibitors and cofactor reagents.

[0082] In an exemplary method, a tagged peptide is appended with N-terminal D- hexahistidine (D-Hise) affinity tags to aid in isolation and purification post-synthesis. This can be achieved using an exemplary method as follows. After successful iterative construction of the desired target sequence by Fmoc-SPPS (see Example 1) on resin and prior to acidolytic cleavage, a further six histidine couplings can be conducted with the Fmoc-D-His(Trt)-OH amino acid to construct on-resin the D-Hise affinity tag. Following acidolytic cleavage from resin, work-up and lyophilization, the D-Hise-tagged peptides can be reconstituted in a buffer system to a desired concentration and applied to a fixed bed of Ni-NTA-functionalized agarose resin, which had been pre-equilibrated in an identical buffer system as used for peptide reconstitution. The resin bed can be flushed with 5 bed volumes of an appropriate buffer (e.g., 10 mM imidazole, 100 mM NaPi, 8 M Urea, pH 8) and the D-Hise-tagged peptides can be subsequently eluted from the Ni-NTA resin through application of an appropriate elution buffer (for example, with 5 mL; e.g, 0.25 M imidazole, 100 mM NaPi, 8 M Urea, pH 4.5). Elutions can be concentrated to a desired volume using a suitable molecular weight cut off spin filter, then dialyzed into an appropriate buffer for downstream assays and quantified using UV-Vis spectrophotometry.

[0083] A diagram of an exemplary workflow for tagged recombinant native protein or tagged recombinant denatured protein purification is shown in Figure 4.EXAMPLESExample 1. Synthesis of affinity tags and tagged peptides or proteins

[0084] Biotinylated L-His-Tag (GSGSHHHHHHGSGS, SEQ ID NO: 7), biotinylated Scrambled-L-His-Tag (HSGHGHSHSGHGSH, SEQ ID NO: 8), biotinylated Retrolnverso-D-His-Tag (sgsghhhhhhsgsg, SEQ ID NO: 9), biotinylated Scrambled-D-His- Tag (hsghgshshghgsh, SEQ ID NO: 10) were ordered through GenScript Biotech (Piscataway, NJ). Peptides were synthesized through Fluorenylmethyloxycarbonyl (Fmoc) protection group chemistry using SPPS.Attomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1

[0085] A general workflow for peptide synthesis was as follows. Fmoc-deprotection was achieved through treatment of the solid-supported peptide with organic solutions of piperidine (most typically 20-40 vol% piperidine in N.N-dimethylformamide (DMF). Following the removal of Fmoc protection group on the resin, the resin was ready for amino acid coupling. A typical coupling cycle could be achieved using any N-terminally protected L- or D-configured (or achiral) amino acid bearing an unprotected (X-carboxylate in equivalents ranging from 1 to 10 equivalents relative to the initially loaded solid- supported amino acid and typically at concentrations ranging from 0.1 to 0.5 mol L1in DMF. Commonly employed coupling reagents include but are not limited to HATU, HBTU, N,N’- diisopropylcarbodiimide (DIC), PyBOP, PyAOP and CITU, and are typically employed in equivalents of 0.95 to 2 equivalents relative to the amino acid to be coupled. Additional racemization suppressants can also be added to typical equivalents of 1 to 4 equivalents relative to the amino acid to be coupled and include, but are not limited to, species such as 1- hydroxy-7-azabenzotriazole (HOAt), 1 -hydroxybenzotriazole (HOBt) or ethyl cyanohydroxyiminoacetate (Oxyma). Addition of base is often required, typically in the form of . -diisopropylethylamine, except in the case of coupling reactions employing DIC and / or Oxyma. Coupling reactions were done at room temperature and coupling durations could vary from 15 min to 120 min, respectively.

[0086] Ninhydrin analysis was undertaken to determine if the amino acid coupling % was satisfactory and the next amino acid in the primary sequence could be added to the growing peptide chain. In general, for a Ninhydrin test a small amount of resin (2 to 5 mg) was removed from the reaction vessel and flow washed in Dichloromethane (DCM) under vacuum until the resin is dry. The dried resin was transferred to a tared tube and the mass was recorded. To the test tube containing the peptide and an empty control tube the following reagents were added: 2 drops of Reagent 1 (76 % w / w phenol in ethanol), 4 drops of Reagent 2 (0.2 mM potassium cyanide KCN in pyridine), and 2 drops of Reagent 3 (0.28 M ninhydrin in ethanol). Both the tubes were covered with Parafilm® and placed in a sand bath, regulated at 95° C for 5 minutes. After removal from the sand bath, 3 mLs of 60 % ethanol in water (v / v) was added to each test tube and centrifuged for 30 seconds. The supernatant was collected, and absorbance of the resin sample was read against the control blank at 570 nm and recorded.

[0087] The % coupling could be determined by using the following equation:% Coupling = { 1 - (NV / SV) } * 100 Equation (1)Attorney Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1

[0088] The NV stands for Ninhydrin Value and gave an estimate for the NH2 group that did not bind to the amino acid in the coupling step. NV could be calculated using the following equation:NV ={(Absorbance5io)*(200)} / resin mass (mg) Equation (2)

[0089] The variables required for NV calculation were obtained from the procedure mentioned above.

[0090] The SV stands for Substitution value and provides the theoretical mole value of the total peptide. SV was calculated using the equation below.New SV = (previous SV) / [1 + (previous SV x AA MW)] Equation (3)

[0091] The previous SV for the first amino acid was determined through the Resin loading value, which was provided by the manufacture. This value indicated the number of [moles / g or mmoles / mg) of resin. While calculating the amino acid molecular weight it was considered that all amino acids have a protection group at the N-terminus and some amino acids also have a side chain protection group. If the % coupling was greater than 99.5 % the next amino acid was coupled to the growing peptide chain. A coupling efficiency of lower than 99.5 % indicated that the coupling has not reached the desired level of completion, hence it was recoupled.

[0092] Following successful coupling of all desired L- and / or D-configured amino acids, including installation of the His-tag (or D-His-tag), the peptide was acidolytically cleaved from resin with one of a variety of trifluoroacetic acid (TFA)-based cleavage cocktails (typically 90:5:5 v / v / v TFAdPrsSiFkfhO), with optional addition of cation scavengers such as thioanisole or thiophenol.

[0093] This crude material was lyophilized and purified by reverse-phase high performance liquid chromatography (RP-HPLC) using a variety of stationary phases, solvent systems and flow parameters. Purified material was finally lyophilized prior to use.Synthesis of His-Tagged Peptides and Proteins

[0094] His-tagged peptides and proteins can be synthetically accessed by solidphase peptide synthesis (SPPS) techniques most typically (but not limited to) employing the Fmoc-protecting group strategy (Fmoc-SPPS), with less common use of the Boc-protecting groups strategy (Boc-SPPS). Such His-tags can vary by location within a peptide chain (primary sequence), composition and tag length. For example, His-tags can readily be incorporated within a synthetically accessed peptide either at the C-terminus, the N-terminus or internally within the primary sequence, and can optionally be separated from the nativeAttorney Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 peptide / protein sequence by a flexible linker region of varying composition [e.g., (GGGGS)n(SEQ ID NO: 11) or (GSGS)n(SEQ ID NO: 12), where n is between 1 and 20], The composition of the His-tag can vary from polyhistidine to histidine residues spaced by non- sterically hindered and conformationally flexible glycine and serine residues [e.g., (HGSHGS)n (SEQ ID NO: 13)]. As shown below, His-tags retain equipotent Ni-binding affinity upon chiral inversion to the enantiomeric D-His-tag - such homochiral D-His-tags are thus considered a further compositional variation of the wider ‘His-tag’ category of metalaffinity tags. His-tags (and D-His-tags) are typically incorporated to a length of between 4 and 10 consecutive histidine residues, most commonly in the form of hexahistidine (Hise / D-Hise).

[0095] The synthesis of His-tagged (and D-His-tagged) peptides and proteins begins through loading of the initial amino acid to a polystyrene (PS)- or PEGA-based solid support (resin), typically crosslinked with 1-2% divinylbenzene (DVB). Loading protocol differs depends on resin functionalization, whereby amino-functionalized resins (e.g., Rink amide, aminomethyl-dimethoxyphenoxyvalearic acid (PAL), Ramage or Sieber resins), providing access to C-terminal amide peptides, are loaded through standard amino acid coupling protocols (see below). Resins providing access to C-terminal acids (e.g., trityl, 2-chlorotrityl, or Wang resins) are loaded through either base-mediated (i.e., use of triethylamine or N,N- diisopropylethylamine) carboxylic acid deprotonation and subsequent displacement at the trityl / triply benzylic center (for trityl and 2-chlorotrityl resins) or through 4- dimethylaminopyridine (DMAP) -catalyzed esterification (for Wang resin). The loaded amino acid (either L- or D-configured) can be, but is not limited to, the C-terminal amino acid of the target sequence or native sequence, or the C-terminal histidine of a C-terminally affixed His- tag (or D-His-tag). After resin loading and quantification through weight or ultraviolet measurement of the piperidine-fulvene adduct following the initial Fmoc-deprotection, peptide extension can be conducted through iterative Fmoc / Boc-deprotection, amino acid coupling and (optionally) acetylation-based capping steps using methods known in the art. In some aspects, in Fmoc-SPPS protocols, Fmoc-deprotection can be achieved through treatment of the solid- supported peptide with organic solutions of piperidine (most typically 20-40 vol% piperidine in N.N-dimethylformamide (DMF)) over periods ranging from 30 seconds to 15 minutes and at temperatures ranging from room temperature to 95 °C, although cooled (e.g., 0 °C) Fmoc-deprotections can also be accomplished on base- sensitive peptide scaffolds. Amino acid coupling cycles can similarly vary in temperature and reaction duration, as well as the chemical identity of the coupling reagent(s), chemical identity of theAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 amino acid to be coupled, addition of base, equivalents of amino acid and coupling reagent(s), and concentration.

[0096] A typical coupling cycle could be achieved using any N-terminally protected L- or D-configured (or achiral) amino acid bearing an unprotected (X-carboxylate in equivalents ranging from 1 to 10 equivalents relative to the initially loaded solid- supported amino acid and typically at concentrations ranging from 0.1 to 0.5 mol L1in DMF. Commonly employed coupling reagents include but are not limited to HATU, HBTU, N,N’- diisopropylcarbodiimide (DIC), PyBOP, PyAOP and CITU, and are typically employed in equivalents of 0.95 to 2 equivalents relative to the amino acid to be coupled. Additional racemization suppressants can also be added to typical equivalents of 1 to 4 equivalents relative to the amino acid to be coupled and include, but are not limited to, species such as 1- hydroxy-7-azabenzotriazole (HOAt), 1 -hydroxybenzotriazole (HOBt) or ethyl cyanohydroxyiminoacetate (Oxyma). Addition of base is often required, typically in the form of . -diisopropylethylamine, except in the case of coupling reactions employing DIC and / or Oxyma. Coupling reaction temperatures and durations can vary from room temperature to 95 °C and 15 min to 120 min, respectively. Following coupling, unreacted (X-amino functionalities can be functionally ‘capped’ through acetylation, typically achieved through treatment with solutions of acetic anhydride in pyridine or basified organic solvent at room temperature for 1-10 min.

[0097] Following successful coupling of all desired L- and / or D-configured amino acids, including installation of the His-tag (or D-His-tag), the peptide is acidolytically cleaved from resin with one of a variety of trifluoroacetic acid (TFA)-based cleavage cocktails (typically 90:5:5 v / v / v TFAdPrsSiFkFFO), with optional addition of cation scavengers such as thioanisole or thiophenol. Acidolytic cleavage is typically performed at room temperature or reduced temperature (e.g., 0 °C) over a period of 1-3 hours, with subsequent filtration of the cleavage solution to remove remnant resin, and concentration of the cleavage solution to afford crude solid peptide material. This crude material is typically lyophilized and purified by reverse-phase high performance liquid chromatography (RP-HPLC) using a variety of stationary phases, solvent systems and flow parameters. Purified material is finally lyophilized prior to use.Attorney Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1Example 2. Properties of affinity tags with amino acid residues in D-enantiomer formMaterials and MethodsAntibody Evasion

[0098] Affinity tags listed in Table 1 were generated in D -enantiomeric form. The D-forms of the tags were then tested in assays to establish whether high-affinity antibodies would bind the retro-inverted (RI) versions of their cognate epitopes. The assay measured the binding of the listed tags to monoclonal and polyclonal antibodies by ELISA.Table 1. Biotinylated affinity tags with amino acids in L-enantiomeric form (shown in capital letters) or D-enantiomeric form (shown in lower case letters).

[0099] The binding of the disclosed affinity tags to their respective antibodies was measured by an enzyme-linked immunosorbent assay (ELISA). Each assay utilized a tagspecific antibody and method steps that were generally similar between the assays. A representative method for the assay is described below for detecting binding to His tags.

[0100] In this example, a biotinylated L-peptide affinity tag containing an internal sequence motif such as HHHHHH (SEQ ID NO: 5) (e.g. Biotin-L-Hise-OOl) is chirally inverted (with or without full sequence reversal) resulting in a new D-peptide tag possessing the internal sequence hhhhhh (SEQ ID NO: 6) (e.g. Biotin-D-RLHise-OOl). Chiral inversion of the tag abrogates binding of the tag to an anti-Hise-tag specific for the L-form the of the peptide. An ELISA plate (Coming™ 3369 HighBind, Corning, NY) is coated with 50 pL of 5 pg / mL recombinant streptavidin solution in PBS. The plate is covered with Sealing Tape (ThermoFisher) and incubated overnight at 4 °C. The plate is emptied, filled with 250 pL of PBST (0.05% v / v Tween-20 in PBS), then aspirated or flicked dry. This is repeated for a total of five washes. Diluted biotinylated peptides (100 pL at 1 pg / mL) are added to the plate and incubated for 1 h at room temperature. The plate is aspirated dry, blocked with 200 pL IX Assay Buffer (ThermoFisher) for 2 h at room temperature, then washed five times with PBST. Primary anti-Hise- antibody (e.g. Abeam, abl8184, mIgG2b) is added at an appropriateAttorney Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 dilution (1 : 103- 107) in Assay Buffer and reacted with the peptides for 2 h, followed by five washes. Secondary peroxidase-conjugated antibody (e.g. KPL anti-mouse IgG 5220-0338) and incubated for 1 h followed by five washes. The plate is aspirated dry, then 100 pL of TMB Substrate (ThermoFisher) is added to the wells. After color development proceeds for several minutes, 100 pL of Stop Solution (ThermoFisher) is added to the wells. The absorbance value of each plate well is measured at 450 nm using a plate spectrometer.Metal binding

[0101] His-tagged proteins exhibit exceptionally high affinities for nickel (Ni2+) ions - a fact exploited for their purification by way of immobilized metal affinity chromatography (IMAC), as is standard in biochemical workflows. Similarly, Ni2+-NTA conjugation to a colorimetric substrate (e.g., horseradish peroxidase) permits the direct detection and quantification of His-tagged proteins.

[0102] Traditionally the Hise tag is installed recombinantly, with only few examples of applications toward chemical peptide synthesis and only in the case of L- peptides. This assay is the first to test metal affinity reagent binding to a synthetically- installed His-tag composed of exclusively D-amino acids (see Table 1).

[0103] For the initial assessment of metal binding affinity, a direct ELISA was performed to detect biotinylated 14-mer peptides containing a Hise tag (GSGSHHHHHHGSGS, SEQ ID NO: 7) using two commercially available Ni2+-NTA-HRP kits (KPL HisDetector™ and Thermo HisProbe™), 1) HisDetector™ Nickel Kit (LGC Clinical Diagnostics) and 2) HisProbe™-HRP Conjugate (ThermoFisher Scientific). In this example, a biotinylated L- or D- peptide affinity tag containing an internal sequence motif such as HHHHHH (SEQ ID NO: 5) (e.g. Biotin-L-His6-001) or hhhhhh (SEQ ID NO: 6) (e.g. Biotin-D-RLHis6-001), is bound to a streptavidin-coated ELISA plate and detected using various Ni-NTA-HRP conjugates.

[0104] An ELISA plate (Corning™ 3369 HighBind, Coming, NY) was coated with 50 pL of 5 pg / mL recombinant streptavidin solution in PBS. The plate was covered with Sealing Tape (ThermoFisher) and incubated overnight at 4 °C. The plate was emptied, filled with 250 pL of PBST (0.05% v / v Tween-20 in PBS), then aspirated with an automated plate washer (BioTeck) or flicked dry. This was repeated for a total of five washes. Diluted biotinylated peptides (100 pL at 1 pg / mL = 0.1 pg / well) were added to the plate and incubated for 1 h at room temperature. The plate was aspirated dry, blocked with 1-200 pL IX Assay Buffer (ThermoFisher) for 2 h at room temperature, then washed 5X with PBST. The Ni-NTA-HRP conjugate (HisDetector™ Nickel-HRP, SeraCare) was diluted in 0.2X orAttomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1IX Assay Buffer at an appropriate dilution (1:1000 - 1:5000) and incubated for 30 min at room temperature, followed by washing. The plate was aspirated dry, then 100 pL of TMB Substrate (ThermoFisher) was added to the wells. After color development proceeds for several minutes, 100 pL of Stop Solution (ThermoFisher) was added to the wells. The absorbance value of each plate well was measured at 450 nm using a plate spectrometer. Results

[0105] From these initial experiments, it was observed that neither monoclonal (shown in Figure 1) nor polyclonal antibodies (not shown) recognized retro-inverted peptide tag sequences.

[0106] Metal binding experiments demonstrated that Ni2+ions bind to the His- tagged peptides, irrespective of a-carbon chirality. In fact, peptides tagged with either D-Hise residues or L-Hise residues bound Ni2+-NTA-HRP with identical affinities (KD = 2.7 nM, Figure 2). The scramble sequences also bound, perhaps due to the presence of 6 intermittent histidine residues within a flexible sequence (GSGSGHSGHGHSHSGHGSH, SEQ ID NO: 2). These results show affinity-based platforms for purifying and analyzing peptides composed entirely of non-canonical amino acids, with unprecedented expediency.Example 3. Design, synthesis, and purification of peptides using disclosed affinity tagsNi-NTA Affinity Chromatography

[0107] Synthetic D-configured anti-VEGF target sequences were appended with N- terminal D-hexahistidine (D-Hise) affinity tags to aid in their isolation and purification postsynthesis. After successful iterative construction of the desired target sequence by Fmoc- SPPS (see Example 1) on resin and prior to acidolytic cleavage, a further six histidine couplings were conducted with the Fmoc-D-His(Trt)-OH amino acid to construct on-resin the D-Hise affinity handle. Following acidolytic cleavage from resin, work-up and lyophilization, crude D-Hise-tagged peptides were reconstituted in an appropriate buffer system (e.g., 0.1 M NaPi, 8 M Urea, 10 mM imidazole pH 8; or 0.1 M NaPi, 6 M Gdn.HCl, 10 mM imidazole, pH 8) and applied at a flow rate of about 1 mL min1over a fixed bed of Ni-NTA-functionalized agarose resin (1 mL) which had been pre-equilibrated in an identical buffer system as used for peptide reconstitution. The resin bed was flushed with 5 - 10 bed volumes (5 - 10 mL) an appropriate buffer (e.g., 0.1 M NaPi, 8 M Urea, 10 mM imidazole, pH 8), further washed with 5 - 10 bed volumes of wash buffer (0.1 M NaPi, 8 M Urea, 20 mM imidazole, pH 6.5) and the D-Hise-tagged peptides subsequently eluted from the Ni-NTA resin through application of an appropriate elution buffer (0.1 M NaPi, 8 M Urea, 250 mM imidazole, pH 4.5). ElutionsAttorney Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1 were dialyzed within a 2 - 3 kDa MWCO membrane into an appropriate buffer for downstream assays, or quantified by UV-Vis spectrophotometry (NanoDrop) and used directly following rapid desalting over a 2 - 3 kDa MWCO spin filter.

[0108] Results for purifying an exemplary synthetic D-configured anti-VEGF target sequence is shown in Figures 3 A and 3B. The peptides prepared and tested for purification in accordance with the described method are listed in Table 2.Table 2. Names and sequences of the synthesized and purified D-configured anti- VEGF target sequences.Example 4. Non-immunogenicity of D-6xHis tag

[0109] Mice are challenged with a series of homochiral and heterochiral peptides or proteins carrying either a D-6xHis tag or an L-6xHis tag (e.g., SEQ. NO 15, SEQ. NO 25, SEQ. NO 26, SEQ. NO 27). The peptides are synthesized using standard SPPS, cleaved under acidic conditions, and purified to homogeneity using preparatory HPLC. Naive adult female BALB / c mice (n = 4 / group) are primed subcutaneously with each peptide (50 pg per injection) emulsified in 100 pL CFA (dO). Whole OVA protein serves as the positive control group. Mice are boosted with peptide (50 pg) emulsified in 100 pL IFA two weeks later (dl4), and serum is recalled on d21 to d28. Antigen- specific IgG from recalled sera is determined by indirect ELISA, with serum from the relevant groups plated against peptide immobilized on a plate. Typically, 100 pL of a 1-10 pg / mL solution of peptide is incubated in a Corning MediumBind ELISA plate for 4 °C overnight. The plate is then washed with 200 pl PBST per well 5X (PBST contains 0.1% Tween in PBS). Each plate is then blocked by the addition of IX Assay Buffer (ThermoFisher Scientific) for 2 h, following by 5X washing with PBST. Serum is then added directly to each well at the appropriate dilution, followed by 5X washing. Peroxidase-labeled anti-mouse IgG (H+L) is then added to each well at a dilution of 1:1000 (100 pL per well at an approximate concentration of 1 pg / mL). After incubation for 1 hr, the plate is washed 5X with PBST. Finally, signal is developed by the addition of 100 pL TMB substrate for 2 - 10 min, with plate development quenched by the addition of Stop Solution (1 N H2SO4 or equivalent). Signal is read out on a plate reader atAttorney Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 450 nm. The AU450results are typically obtained for a single serum dilution of 1:1000, or for a dilution series between LIO1to LIO6. The titer of each response is determined by dilution of the sera from LIO1to 1 : 106, and is taken as the first dilution below with AU450is found to be < 1. In contrast to L-or D-peptides carrying the L-6xHis-tag, D-peptides carrying the D- 6xHis-tag may show low or undetectable levels of signal in the ELISA assay.Table 3. Names and sequences of the polypeptides for the immunogenicity study.INCORPORATION BY REFERENCE

[0110] All US and PCT patent application publications and US patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.EQUIVALENTS

[0111] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W1What is claimed:

1. An affinity tag for protein purification comprising at least one amino acid in D- enantiomer form.

2. The affinity tag of claim 1, wherein the at least one amino acid in D-enantiomer form is a histidine residue in D-enantiomer form.

3. The affinity tag of claim 1 or 2, wherein the affinity tag has substantially no binding to antibodies raised against the affinity tag having all amino acid residues in L- enantiomer form.

4. The affinity tag of any one of claims 1-3 comprising between one and 12 histidine residues in D-enantiomer form.

5. The affinity tag of any one of claims 1-4 comprising between 2 and 3 histidine residues in D-enantiomer form.

6. The affinity tag of any one of claims 1-5 comprising up to 6 histidine residues, wherein at least one histidine residue is in D-enantiomer form; and each histidine residue is flanked by an amino acid that is not a histidine.

7. The affinity tag of claim 6, wherein the amino acid that is not a histidine is selected from the group consisting of a serine and a glycine.

8. The affinity tag of claim 7, wherein the serine residue is in D-enantiomer form.

9. The affinity tag of claim 7, wherein the tag comprises one or more serine residues in L-enantiomer form and one or more serine residues in D-enantiomer form.

10. The affinity tag of any one of claims 1-9, comprising between one and 12 histidine residues in D-enantiomer form and a dissociation constant (KD) from Ni2+ions of between about 0.1 nM and about 1 pM.Attomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W111. The affinity tag of claim 10, wherein the affinity tag has substantially no binding to antibodies raised against an affinity tag comprising six consecutive histidine residues in L-enantiomer form.

12. The affinity tag of any one of claims 1-11, wherein the affinity tag comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 3, 4, or 6.

13. An affinity tag for protein purification, wherein the affinity tag for protein purification comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 or 2 with at least one amino acid residue in D-enantiomer form.

14. A synthetic peptide, comprising the affinity tag of any one of claims 1-13.

15. The synthetic peptide of claim 14, having one or more amino acid mutations relative to the amino acid sequence of a corresponding wild type peptide with all of its amino acid residues in L-enantiomer form.

16. The synthetic peptide of claim 14 or 15, wherein the synthetic peptide and the corresponding wild type peptide with all of its amino acid residues in L-enantiomer form have substantially the same biological function.

17. The synthetic peptide of claim 16, wherein the synthetic peptide is a competitive inhibitor of the corresponding wild type peptide with all of its amino acid residues in L-enantiomer form.

18. The synthetic peptide of claim 17, wherein the synthetic peptide and the corresponding wild type peptide with all of its amino acid residues in L-enantiomer form have substantially the same dissociation constant (KD).

19. The synthetic peptide of any one of claims 14-18, wherein the synthetic peptide is a purified synthetic peptide with about 90% purity.Attomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W120. The synthetic peptide of any one of claims 14-19, wherein the affinity tag is linked to the synthetic peptide via a non-cleavable linker.

21. The synthetic peptide of claim 20, wherein the non-cleavable linker is an amino acid.

22. The synthetic peptide of claim 20, wherein the non-cleavable linker is a polyamino acid.

23. The synthetic peptide of claim 20, wherein the non-cleavable linker is a bifunctional polyethylene glycol (PEG) linker.

24. The synthetic peptide of claim 20, wherein the non-cleavable linker is a heterobifunctional PEG linker.

25. The synthetic peptide of claim 20, wherein the non-cleavable linker is an amino acidfunctional PEG linker.

26. The synthetic peptide of any one of claims 20-25, wherein the affinity tag is selected from the group consisting of biotin, desthiobiotin, azidohomoalanine, homopropargylglycine, and the affinity tag of any one of claims 1-13.

27. The synthetic peptide of any one of claims 14-26, wherein the synthetic peptide comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 14 or 15.

28. A method of producing a tagged peptide or protein, comprising linking a peptide or protein to an affinity tag comprising at least one amino acid in D-enantiomeric form, thereby producing the tagged peptide or protein.

29. The method of claim 28, wherein linking comprises recombinantly producing the peptide or protein with the affinity tag.

30. The method of claim 28, wherein linking comprises chemically linking the affinity tag via a linker to the peptide or protein.Attomey Docket No.: FBZ-00125Flagship Ref. No.: VL83005-W131. The method of any one of claims 28-30, wherein the affinity tag comprises the affinity tag of any one of claims 1-13.

32. The method of claim 30 or 31, wherein the linker comprises a non-cleavable linker.

33. The method of claim 32, wherein the non-cleavable linker is selected from the group consisting of amino acids, polyamino acids, bifunctional polyethylene glycol (PEG) linkers, heterobifunctional PEG linkers, and amino acid-functional PEG linkers.

34. A method of purifying a peptide or protein comprising an affinity tag comprising at least one amino acid in D-enantiomeric form, the method comprising the steps of: a. equilibrating with equilibration buffer a resin having any one of metal ionsCo2+, Ni2+, Cu2+, or Zn2+immobilized in the resin; b. loading the resin with a solution containing the peptide or protein to bind the peptide or protein to the resin; c. washing the resin with wash buffer; and d. eluting bound peptide or protein by passing elution buffer through the resin and collecting the eluate comprising the purified peptide or protein.

35. The method of claim 34, wherein step c) is repeated one or more times.

36. The method of claim 34 or 35, wherein the at least one amino acid in D-enantiomer form is a histidine residue in D-enantiomer form.

37. The method of any one of clams 34-36, wherein the affinity tag has substantially no binding to antibodies raised against the affinity tag having all amino acid residues in L-enantiomer form.

38. The method of any one of clams 34-37 comprising between one and 12 histidine residues in D-enantiomer form.

39. The method of any one of clams 34-38 comprising between 2 and 3 histidine residues in D-enantiomer form.Attomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W140. The method of any one of clams 34-39 comprising up to 6 histidine residues, wherein at least one histidine residue is in D-enantiomer form and wherein each histidine residue is flanked by an amino acid that is not a histidine.

41. The method of claim 40, wherein the amino acid that is not a histidine is selected from the group consisting of a serine and a glycine.

42. The method of claim 41, wherein the serine residue is in D-enantiomer form.

43. The method of claim 41, wherein the serine residue is in L-enantiomer form.

44. The method of any one of claims 34-43 comprising between one and 12 histidine residues in D-enantiomer form and a dissociation constant (KD) from Ni2+ions of between about 0.1 nM and about 1 pM.

45. The method of claim 44, wherein the affinity tag has substantially no binding to antibodies raised against an affinity tag comprising six consecutive histidine residues in L-enantiomer form.

46. The method of claim 45, wherein the affinity tag comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID NO: 3, 4, or 6.

47. A method comprising using the affinity tag of any one of claims 1-13; wherein the method is selected from the group consisting of protein tagging, protein purification, a diagnostic method, a prophylactic method, and a therapeutic method.

48. The affinity tag of claim 12, wherein the affinity tag comprises an amino acid sequence having about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 3, 4, or 6.

49. The affinity tag for protein purification of claim 13, wherein the affinity tag for protein purification comprises an amino acid sequence having about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity,Attomey Docket No.: FBZ-00125 Flagship Ref. No.: VL83005-W1 or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 or 2.

50. The synthetic peptide of claim 27, wherein the synthetic peptide comprises an amino acid sequence having about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 14 or 15.

51. The method of claim 46, wherein the affinity tag comprises an amino acid sequence having about 80% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, or about 95% to about 98% identity to any one of the amino acid sequences set forth in SEQ ID NO: 3, 4, or 6.

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