JP7848139B2 - α-cyclin substrate, method for producing the same, and method for using the same - Google Patents

Description

Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63/026,394, filed on 18 May 2020.
Sequence Listing The sequence listing has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on 18 May 2021 is named Amprion-SUBS-AS-PCT.txt and is 39,523 bytes in size.

Certain degenerative brain diseases, collectively known as "synuclein diseases," involve the pathological accumulation of misfolded α-synuclein (αS) protein in the brains of affected individuals. Misfolded αS protein is an αS protein that possesses a different structural conformation than when it is involved in its typical non-pathogenic, normal function within a biological system. Misfolded αS protein can aggregate and may exist in or as aggregates. Misfolded αS protein may be localized in αS protein aggregates. Misfolded αS protein may be a non-functional protein. Misfolded αS protein may be a pathogenic conformational isomer of the αS protein.

Synuclein diseases include Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA), as well as rare axonal dystrophy. Some evidence suggests that the process of αS misfolding and aggregation can begin years or even decades before the onset of clinical symptoms and substantial brain damage. Therefore, detection of αS aggregates and/or misfolded αS proteins to facilitate early diagnosis of synuclein diseases may prove crucial for enabling intervention before irreversible neuropathological changes occur.

Unfortunately, soluble misfolding αS proteins are present in body fluids in such small amounts that they are extremely difficult to detect. However, significant progress has recently been made in the detection of misfolding αS aggregates (i.e., non-covalent associations of misfolding αS proteins), particularly by seed amplification assays (SAAs) (formerly known as protein misfolding cyclic amplification (PMCA)). See, for example, U.S. Patent No. 20160077111, U.S. Patent No. 20210063416, and U.S. Nonprovisional Patent Application No. 17/154,966, each of which is incorporated herein by reference in whole. Briefly, a biological sample (e.g., blood, skin, cerebrospinal fluid, etc.) is brought into contact with a pre-incubation mixture, which comprises a monomer αS substrate, a buffer composition, a salt, and an indicator to form an incubation mixture. Multiple incubation cycles are performed on the incubation mixture. Each incubation cycle comprises (1) incubating an incubation mixture effective in inducing misfolding and/or aggregation of monomeric αS substrates in the presence of any soluble misfolding αS protein present in the biological sample, and (2) physically disrupting the incubation mixture to "deaggregate," i.e., disintegrate or break down, the misfolding αS aggregates, releasing smaller aggregates. The αS aggregates can then act as "seeds." Detection of misfolding αS aggregates by indicator fluorescence indicates the presence of soluble misfolding αS protein in the biological sample. Figure 1 shows an exemplary depiction of the αS-SAA process using a biological sample containing soluble misfolding αS protein.

A significant limitation of the αS-SAA technology is the difficulty in producing SAA-qualified monomer αS substrates on a scale sufficient for extensive testing. One approach to producing SAA-qualified monomer αS substrates involves transforming intestinal bacterial host cells (e.g., Escherichia coli ("E.Coli")) with an expression vector containing a plasmid represented by a nucleic acid sequence encoding the human αS protein, e.g., SEQ ID NO: 4; culturing the intestinal bacterial host cells under conditions effective for producing the monomer human αS protein; obtaining the human αS protein from the intestinal bacterial host cells; and purifying the human αS protein to obtain recombinant monomer αS substrates. However, the resulting monomer αS substrates sometimes contained "mis-integrated" cysteine instead of tyrosine, particularly at position 136. "Mis-integration" refers to a process in which, during the expression of nucleic acid of a protein from an organism, such as human αS protein, in a host cell, such as a microorganism like Escherichia coli (E. coli), a specific amino acid, such as cysteine, may be unintentionally substituted with another amino acid, such as tyrosine. The presence of a cysteine residue in the monomer αS substrate can lead to self-aggregation and misfolding through dimerization.

αS protein aggregation is a predictable aspect of the pathogenesis of misfolding protein disorders and is advantageously utilized in αS-SAA. However, it is crucial that the monomer αS substrate does not self-aggregate as much as possible. Self-aggregation refers to aggregation that occurs even when soluble misfolding αS protein is not present in the biological sample. The tendency to self-aggregate is an inherent characteristic of αS proteins present in their amino acid sequence.

Depending on their relative dynamics and degree, monomer αS substrate autoaggregation and misfolding can be fatal factors in the use of αS-SAA for amplifying and detecting misfolded αS aggregates in biological samples. When monomer αS substrate autoaggregation occurs, the results may be "false positives," meaning that misfolded αS aggregates are detected even though soluble misfolded αS protein was not present in the biological sample, or that the quantitative assessment of soluble misfolded αS protein in the biological sample is inaccurately high.

Another limitation of existing monomer αS substrates is that they do not aggregate in the presence of soluble misfolded αS protein in the biological sample. This can occur, in particular, when the monomer αS substrate lacks sufficient homology to the soluble misfolded αS protein. If aggregation between the monomer αS substrate and the soluble misfolded αS protein in the biological sample does not occur, the result may be a "false negative," meaning that misfolded αS aggregates are not detected despite the presence of soluble misfolded αS protein in the biological sample, or an inaccurate and low quantitative assessment of soluble misfolded αS protein in the biological sample.

Therefore, on the one hand, the self-aggregation and misfolding of the monomer αS substrate, and on the other hand, the failure to aggregate with soluble misfolded αS proteins in the biological sample, can lead to misdiagnosis or inaccurate prognosis of the tested subject. Thus, a monomer αS substrate is needed that, when used in αS-SAA with appropriate SAA conditions, reduces, slows, or prevents self-aggregation, but still retains its activity in the presence of soluble misfolded αS proteins in the biological sample.

An expression vector is provided for the production of a human αS protein or its conserved variant (ultimately a "monomer αS substrate") that exhibits a reduced tendency to self-aggregate at αS-SAA. The expression vector comprises a nucleic acid sequence encoding the human αS protein or its conserved variant, the nucleic acid sequence containing codons optimized to produce the human αS protein or conserved variant when expressed by an intestinal bacterial host cell such as Escherichia coli (E. coli). In some embodiments, the codons are optimized to avoid amino acid misincorporation. In some embodiments, the codons are optimized to avoid cysteine misincorporation in the expressed protein. In further embodiments, the codons are optimized to avoid cysteine misincorporation at at least one of positions 39, 125, 133, and 136 in the expressed protein.

In one embodiment, the expression vector may be represented by SEQ ID NO: 1 or contain a coding nucleic acid sequence having at least 90% identity with SEQ ID NO: 1. In one embodiment, the expression vector may be a plasmid. For example, the expression vector may be a plasmid that enables protein expression by the T7-lac operon system. In one embodiment, the expression vector may be represented by SEQ ID NO: 2 or be a plasmid containing SEQ ID NO: 1 that has at least 90% identity with SEQ ID NO: 2. In some embodiments, the monomer αS substrate is represented by SEQ ID NO: 6.

A method for producing a purified monomeric human αS substrate or a conserved variant is also provided. The method comprises the steps of transforming a host cell with an expression vector comprising a nucleic acid sequence encoding a monomeric human αS protein or a conserved variant, wherein the nucleic acid sequence comprises codons optimized to avoid misintegration of amino acids in the expressed protein; culturing the host cell under conditions effective for producing a monomeric human αS protein or a conserved variant; obtaining the monomeric human αS protein or a conserved variant from the host cell; and purifying the monomeric human αS protein or a conserved variant by subjecting the human αS protein or a conserved variant to one or more acid precipitation steps at a pH of about 3.5 or less, followed by chromatography, to obtain a purified monomeric human αS substrate or a conserved variant. In some embodiments, the host cell is an intestinal bacterium such as Escherichia coli (E. coli), e.g., E. coli Bl21(DE3), BL21(DE3)-pLysS, etc. In such embodiments, the method further comprises the steps of removing bacterial lipids after purification and/or adding lipopolysaccharide ("LPS") to the monomeric human αS substrate or a conserved variant.

Finally, recombinant enterobacteriaceae cells are provided, each comprising an expression vector for producing human αS protein or a conserved variant, the expression vector comprising a nucleic acid sequence encoding the human αS protein or a conserved variant, the nucleic acid sequence comprising codons optimized to produce the human αS protein or a conserved variant without amino acid misintegration when expressed by the cell.

The present invention can be more easily understood by referring to the following figures.

This figure illustrates an exemplary depiction of a “fast” αS-SAA process using a biological sample containing soluble misfolded αS protein.

This figure shows the gel electrophoresis results of the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3) transformed with the plasmid represented by SEQ ID NO: 2. The results after treatment with dithiothreitol ("DTT") are shown. This figure shows the gel electrophoresis results of the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3) transformed with the plasmid represented by SEQ ID NO: 2. The results without treatment are shown.

This figure shows the gel electrophoresis results of substrates expressed in Escherichia coli (E. coli) strain BL21 (DE3) transformed with the plasmid represented by Sequence ID No. 4, before and after treatment with DTT and filtration with a 30 or 50 kDa filter.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample and purified by acid precipitation to pH 4, using the monomer αS substrate corresponding to SEQ ID NO: 6. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed in a healthy control (HC) using the plasmid represented by SEQ ID NO: 2 and purified by acid precipitation to pH 4, using the monomer αS substrate corresponding to SEQ ID NO: 6.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample and purified by acid precipitation to pH 3.5, using the monomer αS substrate corresponding to SEQ ID NO: 6. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 and purified by acid precipitation to pH 3.5 at HC, using the monomer αS substrate corresponding to SEQ ID NO: 6.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample and purified by acid precipitation to pH 3, using the monomer αS substrate corresponding to SEQ ID NO: 6. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 at HC and purified by acid precipitation to pH 3, using the monomer αS substrate corresponding to SEQ ID NO: 6.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample and purified by acid precipitation to pH 2.5, using the monomer αS substrate corresponding to SEQ ID NO: 6. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 and purified by acid precipitation to pH 2.5, using the monomer αS substrate corresponding to SEQ ID NO: 6.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample and purified by acid precipitation to pH 2.0, using the monomer αS substrate corresponding to SEQ ID NO: 6. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 and purified by acid precipitation to pH 2.0, at HC, using the monomer αS substrate corresponding to SEQ ID NO: 6.

This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3) that was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3) that was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS.

This figure shows the agglutination curves of the αS-SAA "fast assay" (two independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3) using the monomer αS substrate corresponding to SEQ ID NO: 6, which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed MSA sample, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS. This figure shows the agglutination curves of the αS-SAA "fast assay" (two independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed with the plasmid represented by SEQ ID NO: 2 in the presence of an MSA sample, purified by acid precipitation to pH 2.5, and further supplemented with various amounts of LPS.

This flowchart illustrates an exemplary method for purifying monomeric αS substrates that, when used in an αS-SAA assay with appropriate SAA conditions, reduce, slow down, or prevent misfolding and autoaggregation, but retain their activity in the presence of soluble misfolded αS proteins in a biological sample.

Figure 11 shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample, and purified by two acid precipitation steps, first at pH 3.5 and then at pH 2.0, as shown in Figure 11. Figure 11 shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample, and purified by two acid precipitation steps, first at pH 3.5 and then at pH 2.0. Figure 11 shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2 in the presence of a confirmed PD sample, and purified by two acid precipitation steps, first at pH 3.5 and then at pH 2.0, as shown in Figure 11. Figure 11 shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2 and purified by two acid precipitation steps, first at pH 3.5 and then at pH 2.0, as shown in HC.

This figure shows the agglutination curves of the αS-SAA "fast assay" (with three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to approximately pH 3.1, and further purified by a second filtration of the protein, which was dialyzed using a 50 kDa filter in the presence of a synthetic seed. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to approximately pH 3.1, and further purified by a second filtration of the dialyzed protein using a 50 kDa filter in HC. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to approximately pH 3.1, and further purified by a second filtration of the protein, dialyzed using a 30 kDa filter in the presence of a synthetic seed. This figure shows the agglutination curves of the αS-SAA "fast assay" (three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to approximately pH 3.1, and further purified by a second filtration of the dialyzed protein using a 30 kDa filter in HC.

αS Expression Vectors Expression vectors are provided for the production of monomeric αS protein exhibiting reduced tendency to self-aggregate at αS-SAA, which is appropriately purified. The expression vectors comprise a nucleic acid sequence encoding a human αS protein or a conserved variant, the nucleic acid sequence comprising codons optimized to produce a human αS protein or a conserved variant when expressed by a suitable host cell. In one embodiment, the host cell is an intestinal bacterial host cell such as Escherichia coli (E. coli). In another embodiment, the host cell is an S2 insect cell, a yeast cell, Saccharomyces cerevisiae, or Pichia pastoris. In some embodiments, the codons are optimized to avoid amino acid incorporation. In some embodiments, the codons are optimized to avoid cysteine misincorporation in the expressed protein. In a further embodiment, the codon is optimized to avoid misincorporation of cysteine at at least one of the positions 39, 125, 133, and 136 of the expressed protein.

In one embodiment, the expression vector may contain a coding nucleic acid sequence represented by SEQ ID NO: 1 or having at least 90% identity with SEQ ID NO: 1. In one embodiment, the expression vector may be a plasmid. For example, the expression vector may be a plasmid that enables protein expression via the T7-lac operon system. In one embodiment, the expression vector may be a plasmid containing SEQ ID NO: 1, represented by SEQ ID NO: 2 or having at least 90% identity with SEQ ID NO: 2.

Some amino acids can be encoded by two or more codons. A natural hierarchy exists for the specific codons used in certain types of cells. Therefore, a nucleic acid sequence configured for the host cell expression of human αS protein may differ from the corresponding nucleic acid sequence expressing human αS protein in human cells. For example, a nucleic acid sequence configured for the expression of human αS protein in microorganisms such as E. coli may include, for instance, substitutions of typical human codons with typical bacterial codons for selected amino acids. For example, a nucleic acid sequence configured for the expression of human αS protein in microorganisms such as E. coli may include a TAT codon expressing tyrosine instead of a TAC codon, which can lead to the misincorporation of cysteine.

Sequence ID 1, when expressed in Escherichia coli (E. coli), mitigates cysteine misintegration for tyrosine at one or more positions 39, 125, 133, and 136 of Sequence ID 6, compared to the expression of human native nucleic acids in E. coli (e.g., via an expression vector containing a coding nucleic acid sequence including Sequence ID 3, as represented by Sequence ID 4). Cysteine misintegration, in contrast to misintegration of other amino acids, is detectable due to dimerization (see Figure 3). When expressed in recombinant host cells, the produced αS protein (Sequence ID 6) exhibits substantially reduced or no cysteine misintegration at one or more positions 39, 125, 133, and 136.

In various embodiments, expression vectors can be operably ligated to regulatory sequences effective for the expression of an optimized nucleic acid sequence (e.g., Sequence ID No. 1) in intestinal bacterial host cells. The term "operably ligated" refers to the arrangement of various polynucleotide elements relative to each other such that the elements are functionally connected and can interact with one another. Such elements may include, but are not limited to, promoters, enhancers, polyadenylated sequences, one or more introns and/or exons, and the coding sequence of the gene to be expressed. Expression vectors can be operably ligated to regulatory sequences effective for the expression of the nucleic acid sequence represented by Sequence ID No. 1 in Escherichia coli (E. coli). Many suitable vectors are available. Vector components generally include, but are not limited to, origins of replication, one or more marker genes, enhancer elements, promoters, and one or more transcription termination sequences.

The αS protein may be produced recombinantly without modification, or as a fusion polypeptide with a heterologous polypeptide, such as a signal sequence, or with another polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide. Generally, the signal sequence may be a component of the vector, or part of a coding sequence inserted into the vector. In one embodiment, the selected heterologous signal sequence may be recognized and processed by the host cell (i.e., cleaved by a signal peptidase).

Expression vectors typically contain selection genes, also known as selection markers. These selection genes encode proteins necessary for the survival or proliferation of transformed host cells grown in a selection medium. Host cells not transformed with a vector containing selection genes will not survive in culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients unavailable in the complex medium.

Expression vectors contain promoters that are recognized by the host organism and operably ligated to orthogonal protein-coding sequences. Promoters are untranslated sequences located upstream (5') (generally within approximately 100–1000 bp) of the start codon of a structural gene that controls the transcription of the specific nucleic acid sequence to which they are operably ligated. Such promoters are typically classified into two classes: inducible and constitutive. Inducible promoters are those that initiate an increase in transcription levels from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Many promoters recognized by various potential host cells are well known.

In one embodiment, the expression vector may include the coding nucleic acid sequence represented by SEQ ID NO: 1, or a nucleic acid sequence having at least approximately 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 sequences identical to SEQ ID NO: 1. In some embodiments, the nucleic acid sequence includes a sequence having at least 95% identity with SEQ ID NO: 1. The nucleic acid sequence is not SEQ ID NO: 3.

The expression vector may be a plasmid. For example, the expression vector may be the plasmid represented by SEQ ID NO: 2. In some embodiments, the plasmid nucleic acid sequence contains a sequence having at least 95% identity with SEQ ID NO: 2. The plasmid nucleic acid sequence is not SEQ ID NO: 4.

To clarify, Sequence ID No. 1 is an optimized DNA sequence encoding human αS with a C-terminal histogram. Sequence ID No. 1 is part of Sequence ID No. 2 (1459–1899 bp of Sequence ID No. 2). Sequence ID No. 2 is the DNA sequence of the entire vector or plasmid containing Sequence ID No. 1. Sequence ID No. 3 is an unoptimized DNA sequence encoding human αS with a C-terminal histogram. Sequence ID No. 3 is part of Sequence ID No. 4 (1459–1899 bp of Sequence ID No. 4). Sequence ID No. 4 is the DNA sequence of the entire vector or plasmid containing Sequence ID No. 3.

Due to the degenerate nature of the genetic code, a given polypeptide can be encoded using a variety of different nucleotide sequences. This disclosure includes DNA compounds of any sequence encoding the amino acid sequence of the polypeptide described herein. In a similar manner, polypeptides can typically tolerate one or more amino acid substitutions, deletions, and insertions in their amino acid sequence without loss of desired or significant activity.

Nucleotide identity is determined by aligning the residues of two polynucleotides and optimizing the number of identical nucleotides along the length of their sequences. Gaps in either or both sequences are tolerated during alignment to optimize the number of shared nucleotides, but the nucleotides in each sequence must nevertheless remain in their proper order. Preferably, the two nucleotide sequences are compared using the BLASTn program of the BLAST2 search algorithm, as described by Tatusova, et al. (FEMS Microbiology Letters, 174, pp. 247-50 (1999)), which is available on the National Center for Biotechnology Information's website under BLAST in the Molecular Databases section of the World Wide Web. Preferably, the default values for all BLAST2 search parameters are used, including `reward for match = 1`, `penalty for mismatch = -2`, `open gap penalty = 5`, `extension gap penalty = 2`, `gap x dropoff = 50`, `expect = 10`, `wordsize = 11`, and optionally `filter on`. In the comparison of two nucleotide sequences using the BLAST search algorithm, nucleotide identity is referred to as "identity."

Recombinant αS protein-producing cells are also provided. Recombinant enterobacteriaceae cells containing expression vectors for producing human αS protein or a conserved variant are also provided. The expression vector may contain a nucleic acid sequence encoding human αS protein or a conserved variant, and the nucleic acid sequence contains codons optimized to produce human αS protein or a conserved variant when expressed by the cell.

Enterobacteriaceae, more formally known as the family of Gram-negative bacteria, are a family of bacteria belonging to the intestinal flora. Members of the Enterobacteriaceae family are rod-shaped bacteria (bacilli), typically 1–5 μm in length, and usually containing flagella for motility. Examples of intestinal bacteria include Escherichia coli (E. coli), Salmonella, Klebsiella, Shigella, Enterobacter, and Citrobacter.

In some embodiments, the nucleic acid sequence encoding the human αS protein or a conserved variant contains a codon optimized to produce the human αS protein or a conserved variant when expressed by *E. coli* host cells, such as *E. coli* BL21(DE3), *BL21(DE3)-pLysS*, etc. *E. coli* requires expression of the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing for induction of T7 RNA polymerase expression via IPTG or autoinduction. Once T7 RNA polymerase is expressed, it enables transcription of Sequence ID No. 1 in the plasmid. BL21(DE3) cells then possess the desired phenotype.

In various embodiments, the expression vector contained in intestinal bacterial cells may include any of the characteristics of the expression vector described herein. For example, in some embodiments, the expression vector includes a nucleic acid sequence having at least 95% identity with SEQ ID NO: 1. In further embodiments, the recombinant αS protein expressed in recombinant host cells is characterized by the absence or mitigation of amino acid misincorporations, including misincorporation of cysteine at one or more of positions 39, 125, 133, and 136.

The term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant vector has been introduced (a specific target cell and its offspring). Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, the offspring may not actually be identical to the parent cell, but they are still included within the scope of the term “host cell.” Recombinant host cells (e.g., recombinant enterobacteria cells) may be isolated cells, cell lines grown in culture, or cells present in living tissue or organisms.

Method for preparing monomer αS substrates A method for preparing monomer αS substrates for use in αS-SAA is also provided. The method comprises the steps of: providing an intestinal bacterial host cell containing a nucleic acid sequence encoding a human αS protein or a conserved variant, wherein the nucleic acid sequence contains a codon optimized to produce a human αS protein or a conserved variant; culturing the intestinal bacterial host cell under conditions effective for producing a human αS protein or a conserved variant; and obtaining a human αS protein or a conserved variant from the intestinal bacterial host cell. In some embodiments, the method provides an analog or peptide fragment of human αS.

The terms “monomer αS protein” and “monomer αS substrate” are used interchangeably and refer to one or more αS protein molecules or conserved variants of their natural, non-pathogenic configurations. In some embodiments, monomer αS substrates include, essentially consist of, or comprise wild-type or recombinant human αS proteins having 140 amino acids, a molecular weight of 14,460 Da, and represented by the following sequence:

Sequence ID 5:

In some embodiments, the monomer αS protein contains, essentially consists of, or comprises a conserved variant of SEQ ID NO: 5. The conserved variant may be a peptide or amino acid sequence that deviates from SEQ ID NO: 5 only in the substitution or addition of one or more amino acids to amino acids that have similar biochemical properties and have a minimal or beneficial effect on the activity of the resulting protein in αS-SAA. The conserved variant must function substantially similarly to the basic component, i.e., SEQ ID NO: 5. For example, a conserved variant of SEQ ID NO: 5 aggregates with a misfolded αS protein to form aggregates that have substantially the same reaction rate as SEQ ID NO: 5 under similar reaction conditions.

Generally, a conserved variant (of SEQ ID NO: 5 or any of the SEQ ID NOs disclosed herein) may have, for example, one, two, three, four, five, six, seven (5%) and up to fourteen (10%) substitutions, additions, or deletions in the amino acid sequence. In some embodiments, a conserved variant of SEQ ID NO: 5 may include αS proteins from other mammalian species, e.g., rodents and non-human primates. In some embodiments, a conserved variant of SEQ ID NOs: 6-23 may include similarly tagged αS proteins from other mammalian species, e.g., rodents and non-human primates (i.e., the mutation is within the 140-amino acid sequence of the αS protein). In some embodiments, the present invention excludes SEQ ID NO: 5 as a monomer αS substrate.

In some embodiments, the monomer αS substrate comprises a recombinant αS protein containing six additional histidine amino acids (i.e., a polyHis purified tag) at the C-terminus of SEQ ID NO: 5, resulting in a molecular weight of 15,283 Da, represented by the following sequence:

Sequence ID 6:

Therefore, SEQ ID NO: 6 is distinguishable from SEQ ID NO: 5 by six additional histidine amino acids at the C-terminus. Retention of the histidine tag may improve the ability of the human monomer αS substrate to avoid self-aggregation. SEQ ID NO: 6 is further distinguishable from conservative variants of SEQ ID NO: 5 (and SEQ ID NO: 6) in which, for example, one or more amino acids are added to the N-terminus. In some embodiments, conservative variants of SEQ ID NO: 5 with one or more amino acids added to the N-terminus are excluded. However, some embodiments include N-terminal additions. Therefore,

Sequence ID 7:

For example, further refined tags including FLAG, HA, Myc, and V5 are intended, and therefore generate the following sequence numbers.

Sequence ID 8:

Sequence ID 9:

Sequence ID 10:

Sequence ID 11:

Sequence ID 12:

Sequence ID 13:

Sequence ID 14:

Sequence ID 15:

Sequence ID 16:

Sequence ID 17:

Sequence ID 18:

Sequence ID 19:

Sequence ID 20:

Sequence ID 21:

Sequence ID 22:

Sequence ID 23:

The preparation of monomeric αS substrates, polypeptide fragments, mutants, cleaved products, derivatives, and splice variants exhibiting substantially equivalent or altered αS activity compared to wild-type proteins is also intended. These variants may be intentional, for example, obtained by site-directed mutagenesis, or accidental, for example, obtained by mutations in a host that produces α-synuclein protein. The scope of these terms includes the αS proteins specifically enumerated herein, as well as all substantially homologous analogs and allelic variants thereof.

Analogues can be created by conservative amino acid substitutions. “Conservative amino acid substitutions” may include, for example, substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Non-essential amino acid residues are those that can be altered from the wild-type sequence of αS without losing, or more preferably substantially altering, the biological activity of αS. Therefore, predicted non-essential amino acid residues in the αS protein are preferably replaced with other amino acid residues from the same side-chain family. Alternatively, in another embodiment, mutations can be randomly introduced along all or part of the αS coding sequence by saturation mutagenesis, and the resulting mutants can be screened for αS biological activity to identify mutants that retain activity. After mutagenesis of the αS nucleotide sequence, the encoded protein can be recombinantly expressed, and the protein's activity can be determined.

The monomer αS substrates prepared herein exhibit a reduced tendency to self-aggregate. In some embodiments, the monomer αS substrates do not self-aggregate under αS-SAA conditions. In other embodiments, the monomer αS substrates self-aggregate at a much lower level and/or much slower rate than the level or rate of self-aggregation of monomer αS substrates obtained by methods prior to the preparation of the monomer αS substrates. In such embodiments, the rate and/or level of self-aggregation may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or less compared to the level of aggregation of monomer αS substrates obtained using methods other than those described herein. In other embodiments, the monomer αS substrates self-aggregate at a slower rate or lower level than the rate or level of aggregation between the monomer αS substrate and soluble misfolding αS protein. In such cases, αS-SAA detection remains successful because synuclein disease-positive samples can be distinguished from simple autoaggregation of the monomer αS substrate by detection intensity (relative fluorescence units) or the time period during which fluorescence increase begins. The reduction in monomer αS substrate autoaggregation described herein may result from differences in the methods used to prepare the monomer αS substrate, including differences in the resulting monomer αS substrate composition.

A method for preparing a human monomer αS substrate or a conserved variant may involve culturing host cells containing a nucleic acid sequence encoding a human αS protein or a conserved variant, wherein the nucleic acid sequence contains codons optimized to produce the human αS protein or a conserved variant. The cells may be cultured in conventional nutrient media appropriately modified for promoter induction, transformant selection, or amplification of a gene encoding a desired sequence. The culture medium may contain buffer, nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary adjuvants may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be apparent to those skilled in the art. For a discussion of strategies for producing recombinant proteins using Escherichia coli (E. coli), see Gopal G., Kumar A., and Protein J. See 32(6):419-25(2013), the disclosure thereof, which is incorporated herein by reference in its entirety.

A method for preparing the monomer αS substrate may include any of the expression vectors described herein. In some embodiments, the enterobacteria is Escherichia coli (E. coli). In further embodiments, the nucleic acid sequence of the expression vector includes a sequence having at least 95% identity with SEQ ID NO: 1. In yet another embodiment, the expression vector is a plasmid containing a sequence having at least 95% identity with SEQ ID NO: 2. In yet another embodiment, the codons are optimized to avoid amino acid incorporation, including misincorporation of cysteine in the expressed human αS protein or a conserved variant.

After culturing the cells for an appropriate period (e.g., 4–24 hours), the cells are lysed, and the monomer αS protein is purified from the lysed cells. Various methods can be used to lyse the cells, including French press, sonication, freeze-thaw cycles, chemical lysis, and microsolution. Purification may involve various different steps such as centrifugation, column purification, dialysis, and acid precipitation to a pH of approximately 3.5 or lower, optionally followed by the addition of LPS and/or ultrafiltration (10 kDa–300 kDa).

In some embodiments, a method for preparing monomer αS substrates from host cells involves lysing the cells using a microfluidizer. Microfluidizers efficiently disrupt cells while maintaining the integrity of the intracellular contents by supplying a constant, controlled shear rate, resulting in large cell membrane fragments that facilitate subsequent protein purification. The use of microfluidizers for cell lysis can reduce the tendency of purified monomer αS substrates to self-aggregate. An example of a suitable microfluidizer is the LM20 Microfluidizer® High Shear Fluid Homogeneizer manufactured by Microfluidics.

Methods for preparing monomer αS substrates may include a step of separating the monomer αS substrate from other components of the host cell, such as lipids. In some embodiments, obtaining monomer αS substrates from host cells (e.g., Escherichia coli (E. coli)) involves contacting the monomer αS substrate with a lipid removal agent ("LRA") to remove lipid contaminants (i.e., cellular components). Thus, monomer αS substrates mixed with various other cellular components are contacted with LRA after cell lysis, and the monomer αS substrates are removed by centrifugation, which separates proteins from lipids (the LRA and bound lipids proceed to the pellet fraction during centrifugation). The use of LRA to remove undesirable lipid components may improve the ability of the recombinant monomer αS substrate in the resulting composition to avoid self-aggregation. LRA is a commercially available agent based on synthetic calcium silicate hydrate (available from Millipore Sigma).

A method for preparing a monomer αS substrate or monomer αS substrate composition may first include a step of separating the monomer αS substrate from a lipid such as LPS, or from a nucleic acid such as DNA and RNA. In some embodiments, obtaining the monomer αS substrate from a host cell (e.g., Escherichia coli) involves precipitation of the non-synuclein component by adding hydrochloric acid (HCl) to a pH of less than 3.50, including a pH of about 2.0 or lower. In some embodiments, the method includes adding LPS to the acid-precipitated monomer αS substrate.

In one embodiment, obtaining a monomer αS substrate from host cells involves subjecting human αS protein or a conserved variant to one or more acid precipitation steps at a pH of approximately 3.5 or lower, for example, first at pH 3.5 and then again at pH 2, followed by chromatography to obtain a purified monomer αS substrate or conserved variant, thereby precipitation of the non-synuclein component. An example of such an embodiment is shown in the flowchart in Figure 11.

Options for removing contaminants such as metal-binding proteins (e.g., iron uptake regulator proteins; FURs) from monomer αS substrate compositions include iron-IMAC, antibody depletion, genome modification of endogenous Escherichia coli (E. coli) FURs including purification tags (other than histag or other purification tags used by αS proteins), iron saturation to reduce binding to nickel columns, or removal washing with Fe++ in nickel-IMAC. In some embodiments, the purification of the monomer αS substrate involves essentially removing all other proteins (e.g., metal-binding proteins). In further embodiments, the metal-binding protein is an iron uptake regulator (FUR).

αS protein composition The isolated human monomer αS substrate or conserved variant composition may also include a suitable medium for suspending and/or preserving the protein. For example, in some embodiments, the monomer αS substrate or conserved variant composition includes a buffer such as piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or phosphate-buffered saline (PBS). In further embodiments, the composition essentially consists of one of SEQ ID NOs. 5-23 and PIPES. The monomer αS substrate may not substantially contain other substances such as lipid contaminants from lysed intestinal bacterial cells.

In some embodiments, human monomer αS substrates or conserved variant compositions are purified to be essentially free of impurities. Most impurities found in the purified monomer αS substrates or conserved variants are proteins of host cell origin, with only a small amount of potential human-derived impurities. MS/MS detects fragments after enzymatic digestion, and proteins are identified by comparing the fragment footprints to those of known proteins, which are available in known databases. Therefore, some fragments may correspond to microorganisms other than host cells (e.g., Escherichia coli). However, most prokaryotic contaminating proteins will likely originate from host cells.

The relative abundances of all human-derived proteins obtained are extremely low compared to the monomer αS substrates. The most representative human-derived contaminants are cytochrome B5 and keratin-related peptides.

Most bacterial contaminants originate from the intestinal bacterial host cells (e.g., Escherichia coli (E. coli)), which are the expression hosts. To date, the most likely and abundant bacterial contaminant appears to be FUR from Escherichia coli (E. coli). This protein has an affinity for iron and plays a role in regulating the intracellular concentration of iron. When iron binds to FUR, it becomes able to bind to DNA and act as a regulator. Since both iron and nickel are divalent cations, FUR's ability to bind to iron may explain why FUR co-purifies with monomer αS substrates during IMAC purification. αS-SAA competent monomer αS substrates or conserved variants may contain lower concentrations of FUR protein than self-aggregating substrates. A potential mechanism by which FUR induces self-aggregation may involve ionic interactions between the monomer αS substrate and the residual iron or nickel supported by FUR. The molecular weight of FUR is substantially the same as that of αS, and therefore, molecular weight-based identification tools can mask impurities.

Several other notable contaminants from *E. coli* with relatively high abundance and consistent scores include catabolite gene activators, HIT-like proteins, FKBP-type peptidyl-prolyl cis-trans isomerases, and several ribosomal proteins. Catabolite gene activators are another DNA-binding protein, similar to FUR, but do not require metal cofactors. HIT proteins are proteins possessing a histidine triad motif that may be involved in zinc binding and have been shown to be able to bind to nucleotides. Therefore, the triad motif may also have an affinity for nickel, which allows HIT proteins to be co-purified with monomer αS substrates.

Therefore, in some embodiments, the human monomer αS substrate or conserved variant composition essentially contains no other proteins. In some embodiments, the other proteins include metal-binding proteins, and in further embodiments, the metal-binding proteins include FURs. Metal-binding proteins include proteins that bind (e.g., chelate) to metal ions such as sodium, potassium, magnesium, calcium, manganese, iron, cobalt, zinc, nickel, vanadium, molybdenum, and tungsten.

Potential impurities in the human monomer αS substrate or conserved variant composition that are excluded from the composition in embodiments of the present invention may include the following: Iron uptake regulatory protein OS = Escherichia coli; Catabolite gene activator OS = Escherichia coli; 30S ribosomal protein S12 OS = Escherichia coli; HIT-like protein hinT OS = Escherichia coli; FKBP-type peptidyl-prolyl cis-trans isomerase slyD OS = Escherichia coli; Bifunctional polymyxin-resistant protein ArnA OS = Escherichia coli; 50S ribosomal protein L27 OS = Escherichia coli OS = Escherichia coli; Formyltetrahydrofolate defollylase OS = Escherichia coli; 30S ribosomal protein S15 OS = Escherichia coli; Glucosamine-fructose-6-phosphate aminotransferase [isomerized] OS = Escherichia coli; Regulator of Sigma D OS = Escherichia coli; Acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase OS = Escherichia coli; UPF0047 protein yjbQ OS = Escherichia coli; Arabinose 5-phosphate isomerase GutQ OS = Escherichia coli; 50S ribosomal protein L28 OS = Escherichia coli; 30S ribosomal protein S16 OS = Parabacteroides distasonis; 30S ribosomal protein S20 OS = Escherichia coli; arabinose 5-phosphate isomerase KdsD OS = Escherichia coli; 30S ribosomal protein S2 OS = Escherichia coli; 50S ribosomal protein L7/L12 OS = Kineococcus radiotolerance Radiotolerans; Ribosomal RNA large subunit methyltransferase A OS = Escherichia coli; Nucleoside diphosphate kinase OS = Alcanivorax borkumensis; Unclassified HTH-type transcription regulator yeiE OS = Escherichia coli; 30S ribosomal protein S7 OS = Escherichia coli; NADH-quinone oxidoreductase subunit B OS = Geobacter sp; NADH-quinone oxidoreductase subunit B OS = Janaschia sp; 30S ribosomal protein S16 OS = Bacteroides thetaiotaomicron; elongation factor Tu OS = Actinobacillus pleuropneumoniae serotype 3; 50S ribosomal protein L4 OS = Acinetobacter sp.; 50S ribosomal protein L25 OS = Caldicellulosiruptor saccharolisticus; spermidine N(1)-acetyltransferase OS = Escherichia coli; 50S ribosomal protein L4 OS = Meningococcal serum (Neisseria meningitidis) group C; aminoglycoside 3'-phosphotransferase OS = Escherichia coli; presumed unclassified protein yghX OS = Escherichia coli; N-hydroxyarylamine O-acetyltransferase OS = Escherichia coli; outer membrane protein A OS = Escherichia coli; ribosomal RNA small subunit methyltransferase H OS = Thermoanaerobacter tengcongenesis; ribosomal RNA small subunit methyltransferase H OS = Clostridium acetobutyricum; phosphoribosylformylglycineamidinecycloligase OS = Geobacillus sp; unclassified protein yhbW OS = Escherichia coli; putitrous acyl-[acyl-carrier-protein] desaturase desA1 OS = Mycobacterium tuberculosis; riboflavin biosynthesis protein RibD OS = Escherichia coli; ribosomal RNA large subunit methyltransferase G OS = Escherichia coli; bifunctional protein putA OS = Escherichia coli; NADH-quinone oxidoreductase subunit G OS = Escherichia coli; D-amino acid dehydrogenase small subunit OS = Azotobacter vinelandii; peptidase T OS = Erwinia carotovora subsp. atroseptica; cobilic acid A,C-diamide synthase OS = Rhodobacter capsulata; ATP synthase subunit β OS = Rickettsia acali akari); UPF0371 protein M6_Spy1067 OS = Streptococcus pyogene serotype; NAD reducing hydrogenase hoxS subunit α OS = Hydrogen bacteria (Cupriavidus necator); chaperone protein DnaK OS = Escherichia coli; urease subunit α OS = Yersinia enterocolitica serotype; 1-deoxy-D-xylulose-5-phosphate synthase OS = Bordetella avium; translation initiation factor IF-2 OS = Proteus vulgaris.

Methods for using monomer αS substrates in SAA Human monomer αS substrates or conserved variant compositions described herein, which have a reduced tendency to self-aggregate, are useful as substrate proteins for αS-SAA. Exemplary αS-SAA methods include those disclosed in U.S. Patent No. 20160077111 ("Slow Assay"), U.S. Patent No. 20210063416 ("Fast Assay"), and U.S. Nonprovisional Patent Application No. 17/154,966.

Examples are included to more clearly illustrate specific aspects of the present invention.

Example 1: Synthesis of a recombinant monomer αS substrate of SEQ ID NO: 6 using SEQ ID NO: 2 Escherichia coli (E. coli) BL21 (DE3) was transformed with an expression vector containing the plasmid represented by SEQ ID NO: 2, according to the manufacturer's instructions (Lucigen® E. cloni® Express Chemically Competent Cells, MA019 Rev. 31OCT2016). This plasmid contains a codon-optimized nucleic acid sequence represented by SEQ ID NO: 1, which encodes the C-terminal His Tag αS protein represented by SEQ ID NO: 6 without amino acid misincorporation.

Bacterial pellets were grown overnight in-house using self-inducing medium. The pellets were tested for inclusion bodies using B-Per reagent and SDS-PAGE. The αS protein represented by SEQ ID NO: 6 was highly expressed (approximately 30% of total protein), and no inclusion bodies were detected by B-Per. SEQ ID NO: 1 was verified by DNA sequencing.

Example 2: Purification of αS protein by microsolution and acid precipitation A bacterial pellet containing αS protein was prepared as described in Example 1, washed, frozen at -80°C, and stored until use. For purification, the cells were thawed in a water bath set to 30°C for 40-45 minutes in lysis buffer (50 mM NaH₂PO₄ pH: 8.0, 0.3 M NaCl, 0.2 mM EDTA, 20 mM imidazole, 1 mM PMSF, 0.1 mM TCP). The cells were resuspended in a final volume of lysis buffer equivalent to four times the weight of the pellet (80 mL of lysis buffer for 20 g of pellet). The resuspended cells were degassed using a standard vacuum pump. The resuspended cells were lysed using a microfluidizer (LM20 Microfluidizer®). The crude lysate was clarified by centrifugation to remove large cell debris.

The clarified lysate was titrated by gradually adding 1 M HCl while stirring. After reaching the target pH, the acidified lysate was incubated for 20–60 minutes with stirring. The acidified lysate was clarified by centrifugation, and the supernatant was neutralized to pH 8.00 with 1 M NaOH. The neutralized lysate was filtered through a 0.22 μm filter, and the material was packed into a column containing nickel-Sepharose resin.

Chromatography was performed using a standard protocol. After packing the neutralized and filtered lysate, the packed column was washed with a _first wash buffer (50 mM _NaH₂PO₄ pH: 7.4, 0.5 M NaCl, 20 mM imidazole, 0.1 mM TCP) and a _second wash buffer (50 mM _NaH₂PO₄ pH: 7.4, 0.15 M NaCl, 20 mM imidazole, 0.1 mM TCP). The protein was eluted with elution buffer ( ₅₀ mM _NaH₂PO₄ pH: 7.4, 0.15 M NaCl, 250 mM imidazole, 0.1 mM TCP), and the eluted fraction was collected on ice. The eluted fractions were evaluated by SDS-PAGE and Coomassi staining to determine the fraction with the lowest amount of impurities and the highest amount of recombinant αS protein.

The eluted fraction was pooled with high αS and low impurities, then dialyzed in 1×PBS at a dilution factor of 1:200 for 4-5 hours. A second dialysis was performed overnight at a total dilution factor of 1:80,000 (1:400).

The dialyzed material was filtered using a 50,000 Dalton MWCO Amicon centrifuge. The filtered material at 50 kDa was the final αS monomer product. For improved accuracy, the protein concentration was determined by BCA, A280, or a combination of A280 and BCA. The protein was divided into single-use portions, each containing approximately 6.5 mg.

Figures 2A and 2B show the electrophoresis results for the C-terminal His Tag αS protein (SEQ ID NO: 6) prepared using a plasmid (SEQ ID NO: 2) containing the nucleotide sequence (SEQ ID NO: 1). A fixed amount of the C-terminal His Tag αS protein was separated into two fractions. One fraction containing a fixed amount of the C-terminal His Tag αS protein was contacted with DTT under disulfide bond reduction conditions. As shown in Figures 2A and 2B, the reduced and unreduced fractions of the C-terminal His Tag αS protein were essentially identical when the protein amount per lane was 1 μg, 2 μg, and 4 μg, respectively. No bands corresponding to dimerization were observed. Therefore, the plasmid (SEQ ID NO: 2) containing the nucleotide sequence (SEQ ID NO: 1) generates the C-terminal His Tag αS protein sequence (SEQ ID NO: 6) without cysteine misintegration when expressed in Escherichia coli (E. coli).

Comparative Example 1: Gel electrophoresis of recombinant human monomer αS substrate prepared without using optimized codons. Figure 3 shows the electrophoresis results of C-terminal HisTag αS protein (intended to be SEQ ID NO: 6, but likely containing a mixture of SEQ ID NO: 6 and SEQ ID NO: 6-Y136C due to cysteine misincorporation) prepared using a plasmid (SEQ ID NO: 4) containing the nucleotide sequence (SEQ ID NO: 3) before and after DTT treatment. Lane 1 (first lane from the left) shows various molecular weight fractions of the incubation mixture. Lane 2 shows the recombinant monomer αS protein reagent (SD*). Lane 3 shows the filtrate (F) through a 30 kDa cutoff filter. Lane 4 shows the retention solution (R) captured by the 30 kDa cutoff filter. Lane 5 shows the filtrate through a 50 kDa cutoff filter. Lane 6 shows the retention solution captured by the 50 kDa cutoff filter. The C-terminal HisTag αS protein has a nominal molecular weight of approximately 15 kDa, but it migrates slightly higher than the 17 kDa marker in SDS-PAGE. Therefore, electrophoresis of the filtrate through the 30 kDa cutoff filter in lane 3 showed a band of approximately 15 kDa corresponding to the C-terminal HisTag αS protein. In addition to the intended 15 kDa recombinant monomer-folded αS protein, lanes 4, 5, and 6 in Figure 3 showed bands of approximately 36 kDa. After treatment with DTT, the approximately 36 kDa band disappeared, leaving only the expected approximately 15 kDa band, indicating the presence of a dimer and separation into monomers.

Example 3: αS-SAA of acid-precipitated monomer αS substrate
αS-SAA, using acid-precipitated αS monomers, was performed using a "fast assay" procedure with the following parameters:

Figure 4A shows the agglutination curves of the αS-SAA "fast assay" (three independent replicates) in the presence of the confirmed PD sample, where the monomer αS substrate was purified by acid precipitation to pH 4. The monomer αS substrate agglutinated as expected in the presence of the PD sample, reaching Fmax within 60–70 hours. However, the monomer αS substrate showed a high tendency towards autoaggregation when the CSF sample from HC was analyzed (Figure 4B).

Similarly, Figure 5A shows the αS-SAA "fast assay" agglutination curves (three independent replicates) in the presence of the confirmed PD sample, with the monomer αS substrate purified by acid precipitation to pH 3.5. The monomer αS substrate agglutinated as expected in the presence of the PD sample, reaching Fmax between 60 and 70 hours, but exhibiting greater variability than the substrate produced using acid precipitation at pH 4.0. The monomer αS substrate showed moderate auto-aggregation with HC (Figure 5B).

Figure 6A shows the αS-SAA "fast assay" agglutination curves (of three independent replicates) in the presence of a confirmed PD sample, with the monomer αS substrate purified by acid precipitation to pH 3. At pH 3, PD agglutination was as expected in only two of the three wells. The third well showed much lower fluorescence, which can be explained by autoaggregation. More surprisingly, HC (Figure 6B) was "reproducibly positive," indicating that the substrate thus purified is prone to autoaggregation (further supporting the hypothesis that the third replicate from the PD sample may also autoaggregate).

Figure 7A shows the agglutination curves of the αS-SAA "fast assay" (three independent replicates) in the presence of confirmed PD samples, with the monomer αS substrate purified by acid precipitation to pH 2.5. Surprisingly, PD agglutination was significantly affected by low reproducibility, delayed agglutination in positive wells (75–90 hours), and lack of amplification for one replicate, potentially producing false-negative results. Conversely, the monomer αS substrate did not autoaggregate (Figure 7B).

Figures 8A and 8B show the agglutination curves of the αS-SAA "rapid assay" (with three independent repeats) expressed in *E. coli* strain BL21 (DE3), transformed with the plasmid represented by SEQ ID NO: 2 and purified by acid precipitation to pH 2.0, in the presence of a confirmed PD sample (Figure 8A) and in HC (Figure 8B). The results were consistent at pH 2.5, and since only two of the three repeats were positive, there was no autoaggregation in HC, and PD agglutination was low.

Example 4: Addition of LPS to the acid precipitate (pH 2.5) monomer αS substrate prior to αS-SAA for PD samples. LPS was added to the acid precipitate (pH 2.5) monomer αS substrate described in Example 3. Figure 9A shows the agglutination curves (three independent replicates) of the αS-SAA "fast assay" in the presence of a confirmed PD sample using 60,000 endotoxin units (EnU) of LPS. LPS accelerated agglutination, which began at approximately 50 hours. Two replicates were clearly positive and showed reproducible agglutination, while the third showed a much lower maximum fluorescence (Fmax). Despite the variability and low Fmax of one replicate, this sample is considered positive. 60,000 EnU of LPS induced a very high level of autoaggregation (Figure 9B). Addition of 600 EnU of LPS substantially improved the reproducibility of the experiment in the three replicates, all of which were positive at the expected 50–75 hours (Figure 9C). Surprisingly, 600 EnU of LPD did not induce autoaggregation, allowing for clear identification of HC samples as negative (Figure 9D).

Example 5: Addition of LPS to the acid-precipitated (pH 2.5) monomer αS substrate of an MSA sample. LPS was added to the acid-precipitated (pH 2.5) monomer αS substrate. Figures 10A and 10B show the agglutination curves of the (two replicates) αS-SAA "fast assay" in the presence of a confirmed MSA sample, using 600 EnU and 120 EnU, respectively. LPS induced agglutination with low fluorescence, consistent with the MSA diagnosis.

Example 5: Purification of αS protein by micro-solution preparation and double acid precipitation Figure 11 shows a flowchart of an exemplary method for purifying monomeric αS substrates that reduce, slow down, or prevent misfolding and auto-aggregation when used in αS-SAA, but retain their activity in the presence of soluble misfolded αS proteins in a biological sample, along with appropriate αS-SAA conditions.

Therefore, a bacterial pellet containing αS protein was prepared as described in Example 1, washed, frozen at -80°C, and stored until use. For purification, the cells were thawed in a lysis buffer (50 mM NaH₂PO₄ pH: 8.0, 0.3 M NaCl, 0.2 mM EDTA, 20 mM imidazole, 1 mM PMSF, 0.1 mM TCP) in a water bath set at 30°C for 40–45 minutes. The cells were resuspended in a final volume of lysis buffer equivalent to four times the weight of the pellet (80 mL of lysis buffer for 20 g of pellet). The resuspended cells were degassed using a standard vacuum pump. The resuspended cells were lysed using a microfluidizer (LM20 Microfluidizer, Microfluidics®). The crude lysate was clarified by centrifugation to remove large cell debris.

The clarified lysate was titrated by adding 1 M HCl stepwise while stirring. After reaching the target pH of 3.5, the acidified lysate was incubated for 20–60 minutes with stirring. The acidified lysate was clarified by centrifugation. The clarified lysate was titrated again by adding 1 M HCl stepwise while stirring. After reaching the target pH of 2.0, the acidified lysate was incubated for 20–60 minutes with stirring. The double acidified lysate was clarified by centrifugation, filtered through a 0.45 μm filter, and the supernatant was neutralized to pH 8.00 using 1 M NaOH. The neutralized lysate was filtered through a 0.22 μm filter, and the material was packed into a column containing nickel-Sepharose resin.

Chromatography was performed using a standard protocol. After packing the neutralized and filtered lysate, the packed column was washed with a _first wash buffer (50 mM _NaH₂PO₄ pH: 7.4, 0.5 M NaCl, 20 mM imidazole, 0.1 mM TCP) and a _second wash buffer (50 mM _NaH₂PO₄ pH: 7.4, 0.15 M NaCl, 20 mM imidazole, 0.1 mM TCP). The protein was eluted with elution buffer ( ₅₀ mM _NaH₂PO₄ pH: 7.4, 0.15 M NaCl, 250 mM imidazole, 0.1 mM TCP), and the eluted fraction was collected on ice. The eluted fractions were evaluated by SDS-PAGE and Coomassi staining to determine the fraction with the lowest amount of impurities and the highest amount of recombinant αS protein.

The eluted fraction was pooled with high αS and low impurities, then dialyzed in 1×PBS at a dilution factor of 1:200 for 4-5 hours. A second dialysis was performed overnight at a total dilution factor of 1:80,000 (1:400).

The dialyzed material was filtered using a 50,000 Dalton MWCO Amicon centrifuge. The filtered material at 50 kDa was the final αS monomer product. For improved accuracy, the protein concentration was determined by BCA, A280, or a combination of A280 and BCA. The protein was divided into single-use portions, each containing approximately 6.5 mg.

Figures 12A–12D show the αS-SAA "fast assay" agglutination curves (three replicates) of the αS protein obtained in the presence of three different confirmed PD samples (Figures 12A–12C) and HC (Figure 12D). The PD samples showed excellent reproducibility, and HC did not exhibit autoaggregation. This is surprising and counterintuitive, considering the autoaggregation shown in HC using a single acid precipitation step at pH 3.5 (Figure 5B) and the lack of desired agglutination in the PD sample using a single acid precipitation step at pH 2.0 (Figure 8A).

Example 6: Purification of αS protein by microsolution preparation, acid precipitation, and multiple filtration. The αS substrate was prepared as described in Example 2, with the target acid precipitation pH set to approximately 3.1, except that a fixed amount of the dialyzed and filtered αS substrate was filtered twice at 50 kDa, and another fixed amount of the dialyzed and filtered αS substrate was filtered twice at 30 kDa.

Figures 13A–13D show the αS-SAA "fast assay" agglutination curves (for three independent repeats) using the monomer αS substrate corresponding to SEQ ID NO: 6, expressed in Escherichia coli (E. coli) strain BL21 (DE3), which was transformed using the plasmid represented by SEQ ID NO: 2, purified by acid precipitation to approximately pH 3.1, and further purified by a second filtration of the protein after dialyze-filtration using a 50 kDa filter in the presence of a synthetic seed (Figure 13A) and in HC (Figure 13B), or using a 30 kD filter in the presence of a synthetic seed (Figure 13C) and in HC (Figure 13D).

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

Where a range of values is provided, unless explicitly indicated in the context, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated or intervening values within that stated range, are included in the present invention. These smaller upper and lower limits may be independently included within smaller ranges and are included in the present invention, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in the present invention.

The term "approximately" in relation to a number is intended to include ±10% of that number. This applies whether "approximately" changes an independent number or changes a number at either or both ends of a range. In other words, "approximately 10" means 9 to 11. Similarly, "approximately 10 to approximately 20" intends 9 to 22 and 11 to 18. Where the term "approximately" is absent, the exact number is intended. In other words, "10" means 10.

The singular forms "a," "and," and "the" include multiple referents unless explicitly indicated otherwise in the context. Therefore, for example, a reference to "a sample" also includes multiple such samples, and a reference to "a monomeric αS substrate" includes one or more such molecules, etc.

All patents, patent applications, and publications cited herein, as well as the complete disclosure of electronically available materials, are incorporated by reference, whether or not a particular citation herein states so. The above detailed descriptions and examples are given solely for the purpose of clarifying understanding. No unnecessary limitations should be inferred therefrom. The present invention is not limited to the exact details illustrated and described, and variations obvious to those skilled in the art are included within the scope of the invention as defined by the claims.

Sequence Listings 1-23 <223> Synthesis

Claims (11)

A nucleic acid comprising a nucleic acid sequence having at least 95% identity with the nucleic acid sequence represented by Sequence ID No. 1, wherein the nucleic acid sequence encodes a protein containing the amino acid sequence represented by Sequence ID No. 6 when expressed in an intestinal bacterial host cell. An expression vector comprising the nucleic acid described in claim 1. The nucleic acid according to claim 1, wherein the nucleic acid sequence is incorporated into the expression vector such that it contains a nucleic acid sequence having at least 95% identity with the nucleic acid sequence indicated by Sequence ID No. 2. A method for preparing a protein containing the amino acid sequence shown by Sequence ID No. 6,
A step of introducing a nucleic acid containing a nucleic acid sequence having at least 95% identity with the nucleic acid sequence shown by Sequence ID No. 1 into an intestinal bacterial host cell,
A step of culturing the intestinal bacterial host cells under conditions effective for producing the protein,
The process of obtaining the protein from the intestinal bacterial host cells,
Methods that include... The method according to claim 4 , wherein the nucleic acid is incorporated into an expression vector such that it contains a nucleic acid sequence having at least 95% identity with the nucleic acid sequence shown by Sequence ID No. 2. The method according to claim 4 , wherein the intestinal bacteria include Escherichia coli. The method according to claim 4 , further comprising an acid precipitation step for purifying the protein. An intestinal bacterial host cell expressing the nucleic acid described in claim 1. A modified intestinal bacterial host cell expressing a heterologous nucleic acid that has at least 95% identity with the nucleic acid sequence shown by Sequence ID No. 1 and encodes a protein containing the amino acid sequence shown by Sequence ID No. 6. The modified intestinal bacterial host cell according to claim 9 , wherein the intestinal bacteria include Escherichia coli. The nucleic acid according to claim 1, wherein the nucleic acid has codons encoding tyrosine at positions 39 and 125 of the amino acid sequence shown by Sequence ID No. 6, which are TAC.