AU2021273750B2

AU2021273750B2 - Alpha-synuclein substrates and methods for making and using the same

Info

Publication number: AU2021273750B2
Application number: AU2021273750A
Authority: AU
Inventors: Luis CONCHA; Carly FARRIS; Bret HOLGUIN; Yihua Ma
Original assignee: Amprion Inc
Current assignee: Amprion Inc
Priority date: 2020-05-18
Filing date: 2021-05-18
Publication date: 2025-09-18
Anticipated expiration: 2041-05-18
Also published as: AU2021273750A1; CA3178674A1; CN116057382A; JP7848139B2; JP2023526378A; KR20230029638A; WO2021236678A1; IL298225A; EP4154015A4; EP4154015A1; MX2022014319A

Description

WO wo 2021/236678 PCT/US2021/033016

ALPHA-SYNUCLEIN SUBSTRATES AND METHODS FOR MAKING AND USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63/026,394,

filed on May 18, 2020.

SEQUENCE LISTING

[0001] A Sequence Listing has been submitted electronically in ASCII format and is

hereby incorporated by reference in its entirety. The ASCII copy, created on May 18, 2021, is

named Amprion-SUBS-AS-PCT.txt andis Amprion-SUBS-AS-PCT.tx and is39,523 39,523bytes bytesin insize. size.

BACKGROUND

[0002] Certain degenerative brain diseases, collectively termed "synucleinopathies,"

involve pathological accumulation of misfolded alpha-synuclein (aS) proteinin (S) protein inthe thebrain brainof of

affected subjects. A misfolded as protein is S protein is an an Sas protein protein having having a a different different structural structural

conformation than it has when involved in its typical, nonpathogenic normal function within a

biological system. A misfolded as protein may S protein may aggregate aggregate and and may may exist exist in in or or as as an an aggregate. aggregate.

A misfolded as proteinmay S protein maylocalize localizein inan anSas protein protein aggregate. aggregate. A A misfolded misfolded S as protein protein maymay

be a non-functional protein. A misfolded as protein may S protein may be be aa pathogenic pathogenic conformer conformer of of the the SaS

protein.

[0003] Synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies

(DLB), and multiple system atrophy (MSA), as well as rare neuroaxonal dystrophies. Several

lines of evidence indicate that the process of aS misfolding and aggregation may begin years

or decades before the onset of clinical symptoms and substantial brain damage. Thus, detection

of of aS aggregates and/or S aggregates and/ormisfolded as protein misfolded to facilitate S protein early diagnosis to facilitate of synucleinopathies early diagnosis of synucleinopathies

could prove crucial to permit intervention before irreversible neuropathological changes occur.

[0004] Unfortunately, soluble, misfolded as protein is S protein is present present in in such such low low amounts amounts in in

bodily fluids that it is very difficult to detect. Recently, however, significant advances have

been made in the detection of misfolded aS aggregates (i.e., non-covalent associations of

misfolded aS protein), particularly via Seed Amplification Assay (SAA) (formerly known as

protein misfolding cyclic amplification (PMCA)). See, e.g., US20160077111A1,

US20210063416A1, and U.S. Nonprovisional Patent Application No. 17/154,966, each of

which isincorporated which is incorporated by reference by reference herein herein in itsin its entirety. entirety. Briefly, Briefly, a biological a biological sample (e.g., sample (e.g.,

blood, blood, skin, skin, cerebrospinal cerebrospinal fluid, fluid, or or the the like) like) is is contacted contacted with with aa pre-incubation pre-incubation mixture, mixture, the the pre- pre-

incubation mixture comprising a monomeric aS substrate; aa buffer S substrate; buffer composition; composition; aa salt; salt; and and an an

indicator, to form an incubation mixture. Multiple incubation cycles are conducted on the

incubation mixture. Each incubation cycle comprises: (1) incubating the incubation mixture

effective to cause misfolding and/or aggregation of the monomeric aS substrate in the presence

of any soluble, misfolded aS protein present in the biological sample, and (2) physically

disrupting the incubation mixture to "de-aggregate," i.e., break up or disrupt, the misfolded as S

aggregates to release smaller aggregates. The aS aggregates may then act as "seeds."

Detection Detection of of misfolded misfolded as aggregate via S aggregate via indicator indicator fluorescence fluorescence indicates indicates the the presence presence of of

soluble, misfolded as protein in S protein in the the biological biological sample. sample. An An example example depiction depiction of of the the S-SAA aS-SAA

process with a biological sample containing soluble, misfolded as protein is S protein is shown shown in in Figure Figure

1. 1.

[0005] A significant limitation to aS-SAA technology is S-SAA technology is the the difficulty difficulty of of producing producing SAA- SAA-

competent monomeric as substrate at S substrate at aa sufficient sufficient scale scale for for widespread widespread testing. testing. One One approach approach

to producing SAA-competent monomeric aS substrate has S substrate has been been to to transform transform into into an an

enterobacterial host cell (e.g., Escherichia Coli ("E. Coli")) an expression vector comprising

a nucleic acid sequence coding for human aS protein, such as the plasmid represented by SEQ

ID NO: 4; culture the enterobacterial host cell under conditions effective to produce the monomeric human aS protein; obtain the human as protein from S protein from the the enterobacterial enterobacterial host host cell; cell; and purify the human as protein to S protein to yield yield recombinant recombinant monomeric monomeric SaS substrate. substrate. However, However, the the resultant monomeric as substratehas, S substrate has,in insome someinstances, instances,contained contained"misincorporated" "misincorporated"cysteine cysteine in place of tyrosine, especially at position 136. "Misincorporation" refers to the process by which certain amino acids, e.g., cysteine, may be unintentionally substituted for other amino acids, e.g., tyrosine, when expressing a nucleic acid for a protein of one organism, e.g., a human nucleic acid encoding human aS protein, in a host cell, e.g., a microorganism such as E. Coli.

The presence of cysteine residues in monomeric as substrate can S substrate can lead lead to to self-aggregation self-aggregation and and

misfolding by dimerization.

[0006] While aggregation of aS protein is S protein is an an expected expected part part of of the the pathology pathology of of misfolded misfolded

protein disorders and is used to advantage in aS-SAA, itis S-SAA, it iscrucial crucialfor formonomeric monomericSas substrate substrate

to self-aggregate as little as possible. Self-aggregation refers to aggregation that would occur

even in the absence of soluble, misfolded aS protein in S protein in the the biological biological sample. sample. The The propensity propensity

to self-aggregate is an intrinsic characteristic of aS protein that S protein that resides resides in in its its amino amino acid acid

sequence.

[0007] Depending on the relative kinetics and extent, self-aggregation and misfolding of

the monomeric as substrate can S substrate can prove prove to to be be aa fatal fatal factor factor in in the the use use of of S-SAA aS-SAA toto amplify amplify and and

detect misfolded as aggregate in S aggregate in aa biological biological sample. sample. When When self-aggregation self-aggregation of of the the

monomeric as substrate occurs, S substrate occurs, the the result result may may be be aa "false "false positive"-that positive"-that is, is, misfolded misfolded Sas

aggregate is detected notwithstanding that no soluble, misfolded as aS protein was present in the

biological sample-or an inaccurately high quantitative assessment of soluble, misfolded aS

protein in the biological sample.

[0008] Another limitation of existing monomeric as substrates is S substrates is the the failure failure of of the the

monomeric as substrate to S substrate to aggregate aggregate in in the the presence presence of of soluble, soluble, misfolded misfolded aS aS protein protein in in the the

PCT/US2021/033016

biological sample. This can occur, among other times, when the monomeric as substrate lacks S substrate lacks

sufficient homology with the soluble, misfolded aS protein. When aggregation of the

monomeric aS substrate and S substrate and soluble, soluble, misfolded misfolded aS aS protein protein in in the the biological biological sample sample do do not not

occur, the result may be a "false negative"-that is, misfolded as aggregate is S aggregate is not not detected detected

notwithstanding that soluble, misfolded aS protein was present in the biological sample-or

an inaccurately low quantitative assessment of soluble, misfolded aS protein in the biological

sample.

[0009] Thus, self-aggregation and misfolding of the monomeric as substrate on S substrate on the the one one

hand, and a failure to aggregate with soluble, misfolded aS protein in the biological sample on

the other, can result in misdiagnosis or inaccurate prognosis of tested subjects. Accordingly,

there there is isa aneed forfor need monomeric as substrate monomeric that, that, S substrate together with proper together with SAA conditions, proper reduces, SAA conditions, reduces,

slows, or prevents self-aggregation when used in aS-SAA, yet retains S-SAA, yet retains its its activity activity in in the the

presence of soluble, misfolded aS protein in a biological sample.

SUMMARY

[0010] An expression vector is provided for production of human aS protein or a

conservative variant thereof (ultimately "monomeric as substrate") that S substrate") that exhibits exhibits aa decreased decreased

tendency tendencytotoself-aggregate in aS-SAA. self-aggregate The The in S-SAA. expression vectorvector expression comprises a nucleic comprises acid a nucleic acid

sequence coding for human aS protein or a conservative variant thereof, the nucleic acid

sequence sequencecomprising comprisingcodons thatthat codons have have been been optimized to produce optimized human ashuman to produce protein or a S protein or a

conservative variant when expressed by an enterobacterial host cell such as E. Coli. In some

aspects, the codons have been optimized to avoid amino acid misincorporation. In some

aspects, the codons have been optimized to avoid cysteine misincorporation in the expressed

protein. protein.InInfurther aspects, further the codons aspects, have been the codons haveoptimized to avoid to been optimized cysteine avoid misincorporation cysteine misincorporation

in at least one of positions 39, 125, 133, and 136 of the expressed protein.

PCT/US2021/033016

[0011] In one aspect, the expression vector may comprise a coding nucleic acid sequence

represented by SEQ ID NO: 1 or having at least 90% identity with SEQ ID NO: 1. In one

aspect, the expression vector may be a plasmid. For example, the expression vector may be a

plasmid that enables the expression of a protein by means of the T7-lac operon system. In one

aspect, the expression vector may be a plasmid comprising SEQ ID NO: 1, as represented by

SEQ ID NO: 2 or having at least 90% identity with SEQ ID NO: 2. In some aspects, the

monomeric monomeric aS aS substrate substrate is is represented represented by by SEQ SEQ ID ID NO: NO: 6. 6.

[0012] Also provided is a method for making a purified monomeric human aS substrate or

a conservative variant. The method comprises transforming into a host cell an expression

vector comprising a nucleic acid sequence coding for monomeric human aS protein or a

conservative variant, the nucleic acid sequence comprising codons that have been optimized to

avoid amino acid misincorporation in the expressed protein; culturing the host cell under

conditions effective to produce the monomeric human aS protein or a conservative variant;

obtaining the monomeric human aS protein or S protein or aa conservative conservative variant variant from from the the host host cell; cell; and and

purifying the monomeric human as protein or S protein or aa conservative conservative variant variant by by subjecting subjecting the the human human

as protein or S protein or aa conservative conservative variant variant to to one one or or more more acid acid precipitation precipitation steps steps at at aa pH pH of of about about

3.5 or less, followed by chromatography, to yield the purified monomeric human aS substrate

or a conservative variant. In some aspects, the host cell is an enterobacteria such as E. Coli,

e.g., E. Coli B121 (DE3), BL21(DE3)-pLysS, B121(DE3), BL21(DE3)-pLysS, and and the the like. like. In In such such aspects, aspects, the the method method further further

comprises eliminating bacterial lipids and/or adding lipopolysaccharide ("LPS") to the

monomeric human as aS substrate or a conservative variant after purification.

[0013] Finally, a recombinant enterobacterial cell is provided, the recombinant

enterobacterial cell comprising an expression vector for production of human aS protein or a

conservative variant, the expression vector comprising a nucleic acid sequence coding for

human aS protein or a conservative variant, the nucleic acid sequence comprising codons that have been optimized to produce human as protein or S protein or aa conservative conservative variant variant without without amino amino acid misincorporation when expressed by the cell.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The present invention may be more readily understood by reference to the following

Figures, wherein:

[0015] aS-SAAprocess Figure 1 is an example depiction of the "fast" S-SAA processusing usingaabiological biological

sample that contains soluble, misfolded as protein. S protein.

[0016] assubstrate Figures 2A and 2B show gel electrophoresis results of the monomeric S substrate

corresponding to SEQ ID NO: 6 expressed in E. coli strain BL21 (DE3)transformed BL21(DE3) transformedusing usingthe the

plasmid represented by SEQ ID NO: 2, wherein Figure 2A shows the results upon treatment

with dithiothreitol ("DTT"), and Figure 2B shows the results without treatment.

[0017] Figure 3 shows gel electrophoresis results of the substrate expressed in E. coli strain

BL21 (DE3) transformed BL21(DE3) transformed using using the the plasmid plasmid represented represented by by SEQ SEQ ID ID NO: NO: 44 before before and and after after

treatment with DTT and filtrations with 30 or 50kDa filters.

[0018] Figure 4A and Figure 4B show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (ofthree three

replicates individually) using the monomeric as substrate corresponding S substrate corresponding to to SEQ SEQ ID ID NO: NO: 66

expressed in E. coli strain BL21 (DE3) transformed BL21(DE3) transformed using using the the plasmid plasmid represented represented by by SEQ SEQ ID ID

NO: 2, purified by acid precipitation to pH 4, in the presence of a confirmed PD sample (Figure

4A) and in a healthy control (HC) (Figure 4B).

[0019] Figure 5A and Figure 5B show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (ofthree three

expressed in E. coli strain BL21 (DE3)transformed BL21(DE3) transformedusing usingthe theplasmid plasmidrepresented representedby bySEQ SEQID ID

NO: 2, purified by acid precipitation to pH 3.5, in the presence of a confirmed PD sample

(Figure 5A) and in an HC (Figure 5B).

[0020] aS-SAA"fast Figure 6A and Figure 6B show S-SAA "fastassay" assay"aggregation aggregationcurves curves(of (ofthree three

replicates individually) using the monomeric as substratecorresponding S substrate correspondingto toSEQ SEQID IDNO: NO:66

expressed in E. coli strain BL21 (DE3) transformed using the plasmid represented by SEQ ID

NO: 2, purified by acid precipitation to pH 3, in the presence of a confirmed PD sample (Figure

6A) and in an HC (Figure 6B).

[0021] Figure 7A and Figure 7B show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (ofthree three

NO: 2, purified by acid precipitation to pH 2.5, in the presence of a confirmed PD sample

(Figure 7A) and in an HC (Figure 7B).

[0022] Figure 8A and Figure 8B show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (ofthree three

NO: 2, purified by acid precipitation to pH 2.0, in the presence of a confirmed PD sample

(Figure 8A) and in an HC (Figure 8B).

[0023] aS-SAA"fast Figures 9A-9D show S-SAA "fastassay" assay"aggregation aggregationcurves curves(of (ofthree threereplicates replicates

individually) using the monomeric as substrate corresponding S substrate corresponding to to SEQ SEQ ID ID NO: NO: 66 expressed expressed in in

E. coli strain BL21 (DE3) transformed using the plasmid represented by SEQ ID NO: 2, purified

by acid precipitation to pH 2.5 and further supplemented with varying amounts of LPS, in the

presence of confirmed PD samples (Figures 9A and 9C) and in HCs (Figure 9B and 9D).

[0024] Figure 10A and Figure 10B show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (oftwo two

NO: 2, purified by acid precipitation to pH 2.5 and further supplemented with varying amounts

of LPS, in the presence of confirmed MSA samples.

[0025] aS Figure 11 shows a flowchart of an example method for purifying monomeric S

substrate that, together with proper SAA conditions, reduces, slows, or prevents altogether

misfolding and self-aggregation when used in an aS-SAA assay, yet S-SAA assay, yet retains retains its its activity activity in in the the

presence of soluble, misfolded aS protein in a biological sample.

[0026] Figures 12A-12D show aS-SAA "fastassay" S-SAA "fast assay"aggregation aggregationcurves curves(of (ofthree three

NO: 2, purified by two acid precipitation steps - first at pH 3.5 and again at pH 2.0, as depicted

in Figure 11 - in the presence of confirmed PD samples (Figures 12A-12C) and in an HC

(Figure 12D).

[0027] aS-SAA"fast Figures 13A-13D show S-SAA "fastassay" assay"aggregation aggregationcurves curves(of (ofthree three

NO: 2, purified by acid precipitation to pH about 3.1, and further purified by a second filtration

of the dialyzed, filtered protein using a 50kDa filter in the presence of synthetic seeds (Figure

13A) and in an HC (Figure 13B) or using a 30kDa filter in the presence of synthetic

seeds (Figure 13C) and in an HC (Figure 13D).

DETAILED DESCRIPTION

aS Expression Vector S Expression Vector

[0028] An expression vector is provided for production of monomeric as protein that, S protein that,

properly purified, exhibits a decreased tendency to self-aggregate in aS-SAA. Theexpression S-SAA. The expression

vector comprises a nucleic acid sequence coding for human aS protein or a conservative

variant, the nucleic acid sequence comprising codons that have been optimized to produce

human aS proteinor S protein oraaconservative conservativevariant variantwhen whenexpressed expressedby byaasuitable suitablehost hostcell. cell.In Inone one aspect, aspect, the the host host cell cell is is an an enterobacterial enterobacterial host host cell cell such such as as E. E. Coli. Coli. In In another another aspect aspect the the host host cell is an S2 insect cell, a yeast cell, Saccharomyces cerevisiae, or Pichia pastoris. In some aspects, the codons have been optimized to avoid amino acid incorporation. In some aspects, the codons have been optimized to avoid cysteine misincorporation in the expressed protein.

In further aspects, the codons have been optimized to avoid cysteine misincorporation in at

least one of positions 39, 125, 133, and 136 of the expressed protein.

[0029] In one aspect, the expression vector may comprise a coding nucleic acid sequence

SEQ ID NO: 2 or having at least 90% identity with SEQ ID NO: 2.

[0030] Some amino acids can be coded for by more than one codon. A natural hierarchy

exists for certain codons to be used in certain types of cells. Thus, a nucleic acid sequence

configured for host cell expression of human aS protein may be different from a corresponding

nucleic acid sequence that expresses human aS protein in human cells. For example, a nucleic

acid sequence configured for expression of human aS protein in a microorganism such as E.

Coli, may include, e.g., substitution of bacteria-typical codons for human-typical codons for

selected amino acids. For example, the nucleic acid sequence configured for expression of

human aS protein in a microorganism such as E. Coli may include a TAT codon expressing

tyrosine rather than a TAC codon, which can lead to cysteine misincorporation.

[0031] SEQ ID NO: 1, upon expression in E. Coli, mitigates misincorporation of, among

other amino acids, cysteine for tyrosine at one or more of positions 39, 125, 133, and 136 in

SEQ ID NO: 6 compared to expression of human native nucleic acid (e.g., via an expression

vector comprising a coding nucleic acid sequence comprising SEQ ID NO: 3, as represented by SEQ ID NO: 4) in E. Coli. Cysteine misincorporation, as opposed to misincorporation of other amino acids, is detectable because of the formation of dimers (see Figure 3). Upon expression in the recombinant host cell, the as protein (SEQ S protein (SEQ ID ID NO: NO: 6) 6) being being produced produced has has substantially decreased or no cysteine misincorporation at one or more of positions 39, 125,

133, and 136.

[0032] In various aspects, the expression vector may be operatively linked to regulatory

sequences effective for expression of an optimized nucleic acid sequence (e.g., SEQ ID NO:

1) in the enterobacterial host cell. The term "operably linked" refers to the arrangement of

various polynucleotide various polynucleotideelements relative elements to each relative toother each such that other the that such elements the are functionally elements are functionally

connected and can interact with each other. Such elements may include, without limitation, a

promoter, an enhancer, a polyadenylation sequence, one or more introns and/or exons, and a

coding sequence of a gene of interest to be expressed. The expression vector may be

operatively linked to regulatory sequences effective for expression of the nucleic acid sequence

represented by SEQ ID NO: 1 in E. Coli. Many suitable vectors are available. The vector

components generally include, but are not limited to, one or more of the following: an origin

of replication, one or more marker genes, an enhancer element, a promoter, and a transcription

termination sequence.

[0033] The as protein may S protein may be be recombinantly recombinantly produced produced without without modification modification or or may may be be

produced as a fusion polypeptide with a heterologous polypeptide, e.g., a signal sequence or

other polypeptide having a specific cleavage site at the N-terminus of the mature protein or

polypeptide. In general, the signal sequence may be a component of the vector, or it may be a

part of the coding sequence that is inserted into the vector. In one aspect, the heterologous

signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal

peptidase) by the host cell.

[0034] Expression vectors usually contain a selection gene, also termed a selectable

marker. The selection gene encodes a protein necessary for the survival or growth of

transformed host cells grown in a selective culture medium. Host cells not transformed with

the vector containing the selection gene will not survive in the culture medium. Typical

selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, e.g.,

ampicillin, neomycin, methotrexate, or tetracycline; (b) complement auxotrophic deficiencies;

or (c) supply critical nutrients not available from complex media.

[0035] Expression vectors will contain a promoter that is recognized by the host organism

and is operably linked to an orthogonal protein coding sequence. Promoters are untranslated

sequences located upstream (5') to the start codon of a structural gene (generally within about

100 to 1000 bp) that control the transcription of the particular nucleic acid sequence to which

they are operably linked. Such promoters typically fall into two classes: inducible and

constitutive. Inducible promoters are promoters that initiate increased levels of transcription

from DNA under their control in response to some change in culture conditions, e.g., the

presence or absence of a nutrient or a change in temperature. Many promoters recognized by

a variety of potential host cells are well known.

[0036] In one aspect, the expression vector may comprise a coding nucleic acid sequence

represented by SEQ ID NO: 1 or a nucleic acid sequence having a sequence identity percentage

to SEQ ID NO: 1 of at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99. In some aspects,

the nucleic acid sequence comprises a sequence having at least 95% identity with SEQ ID NO:

1. The nucleic acid sequence is not SEQ ID NO: 3.

[0037] The expression vector may be a plasmid. For example, the expression vector may

be a plasmid represented by SEQ ID NO: 2. In some aspects, the plasmid nucleic acid sequence

comprises a sequence having at least 95% identity with SEQ ID NO: 2. The plasmid nucleic

acid sequence is not SEQ ID NO: 4.

[0038] To be clear, SEQ ID NO: 1 is the optimized DNA sequence that encodes for human

aS with a C-terminal histag. SEQ ID NO: 1 is a part of SEQ ID NO: 2 (from 1459 to 1899bp

of SEQ ID NO: 2). SEQ ID NO: 2 is the DNA sequence of the whole vector or plasmid

including SEQ ID NO: 1. SEQ ID NO: 3 is the non-optimized DNA sequence that encodes for

human aS with a C-terminal histag. SEQ ID NO: 3 is part of SEQ ID NO: 4 (from 1459 to

1899bp of SEQ ID NO: 4). SEQ ID NO: 4 is the DNA sequence of the whole vector or plasmid

including SEQ ID NO: 3.

[0039] Due to the Due to thedegenerate degenerate nature nature of genetic of the the genetic code, code, a a variety variety of different of different nucleotide nucleotide

sequences can be used to encode a given polypeptide. The present disclosure includes DNA

compounds of any sequence that encode the amino acid sequences of the polypeptides

described herein. In similar fashion, a polypeptide can typically tolerate one or more amino

acid substitutions, deletions, and insertions in its amino acid sequence without loss or

significant loss of a desired activity.

[0040] Nucleotide identity is determined by aligning the residues of the two

polynucleotides to optimize the number of identical nucleotides along the lengths of their

sequences. Gaps in either or both sequences are permitted in making the alignment in order to

optimize the number of shared nucleotides, although the nucleotides in each sequence must

nonetheless remain in their proper order. Preferably, two nucleotide sequences are compared

using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al.

(FEMS Microbiology Letters, 174, p. 247-50 (1999)), and available on the world wide web at

the National Center for Biotechnology Information website, under BLAST in the Molecular

Database section. Preferably, the default values for all BLAST 2 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 "identities."

Recombinant RecombinantasS Protein-Producing Protein-ProducingCells Cells

[0041] A recombinant enterobacterial cell is also provided, wherein the cell comprises an

expression vector for production of human aS protein or a conservative variant. The expression

vector may comprise a nucleic acid sequence coding for human aS protein or a conservative

human as protein or S protein or aa conservative conservative variant variant when when expressed expressed by by the the cell. cell.

[0042] Enterobacteria, more formally known as Enterobacteriaceae, are a family of Gram-

negative bacteria. Members of the Enterobacteriaceae family are bacilli (rod-shaped), are

typically 1-5 um µm in length, and usually include flagella for movement. Examples of

enterobacteria include E. Coli, Salmonella, Klebsiella, Shigella, Enterobacter, and Citrobacter.

[0043] In some aspects, the nucleic acid sequence coding for human aS protein or a

conservative variant comprises codons that have been optimized to produce human as protein S protein

or a conservative variant when expressed by an E. Coli host cell, e.g., E. Coli B121 (DE3), B121(DE3),

BL21(DE3)-pLysS, and the like. E. coli needs to express the gene for T7 RNA polymerase

under control of a lacUV5 promoter, allowing expression of the T7 RNA polymerase to be

induced with IPTG or by autoinduction. Once T7 RNA polymerase is expressed, it enables the

transcription of SEQ ID NO: 1 in the plasmid. BL21(DE3) cells have the required phenotype.

[0044] In various aspects, the expression vector included in the enterobacterial cell may

include any of the features for an expression vector described herein. For example, in some

aspects, the expression vector comprises a nucleic acid sequence having at least 95% identity

with SEQ ID NO: 1. In further aspects, the recombinant as aS protein expressed in the

recombinant host cell is characterized by the absence of or mitigation of amino acid

WO wo 2021/236678 PCT/US2021/033016

misincorporation, misincorporation, including including cysteine cysteine misincorporation misincorporation at at one one or or more more of of positions positions 39, 39, 125, 125, 133, 133,

and 136.

[0045] The term "recombinant host cell" (or simply "host cell") refers to a cell (the

particular subject cell and the progeny of such a cell) into which a recombinant vector has been

introduced. Because certain modifications may occur in succeeding generations due either to

mutation or environmental influences, the progeny may not, in fact, be identical to the parent

cell, but are still included within the scope of the term "host cell." A recombinant host cell

(e.g., a recombinant enterobacterial cell) may be an isolated cell, a cell line grown in culture,

or may be a cell that resides in a living tissue or organism.

Methods Methodsfor forPreparing Monomeric Preparing as Substrate Monomeric S Substrate

[0046] A method for preparing monomeric aS substrate for S substrate for use use in in S-SAA aS-SAA isis also also

provided. The method comprises providing an enterobacterial host cell comprising a nucleic

acid sequence coding for human aS protein or a conservative variant, the nucleic acid sequence

comprising codons that have been optimized to produce human as protein or S protein or aa conservative conservative

variant; culturing the enterobacterial host cell under conditions effective to produce the human

aS protein or a conservative variant; and obtaining the human as protein or S protein or aa conservative conservative

variant from the enterobacterial host cell. In some aspects, the method provides analogs or

peptide fragments of human aS. S.

[0047] The phrases "monomeric as protein" and S protein" and "monomeric "monomeric SaS substrate" substrate" are are used used

interchangeably and refer to one or more aS protein molecules S protein molecules or or aa conservative conservative variant variant in in

their native, nonpathogenic configuration. In some aspects, the monomeric as substrate S substrate

comprises, consists essentially of, or consists of wildtype or recombinant human as protein S protein

having 140 amino acids, having a molecular mass of 14,460 Da, and being represented by the

sequence:

[0048] SEQ ID NO: 5:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0049] In some aspects, the monomeric aS protein comprises, S protein comprises, consists consists essentially essentially of, of, or or

consists of a conservative variant of SEQ ID NO: 5. A conservative 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 several amino acids for amino acids having similar biochemical properties and having

a minimal or beneficial impact on the activity of the resultant protein in the aS-SAA. S-SAA. AA

conservative variant must functionally perform substantially like the base component, i.e., SEQ

ID NO: 5. For example, a conservative variant of SEQ ID NO: 5 will aggregate with misfolded

as protein and S protein and will will form form aggregates aggregates with with substantially substantially similar similar reaction reaction kinetics kinetics as as SEQ SEQ ID ID NO: NO:

5 under similar reaction conditions.

[0050] Generally, a conservative variant (of SEQ ID NO: 5 or of any of the SEQ ID NOs

disclosed herein) may have for example, one, two, three, four, five, six, seven (5%), and up to

14 (10%) substitutions, additions, or deletions in the amino acid sequence. In some aspects,

the conservative variant of SEQ ID NO: 5 may include as protein of S protein of other other mammalian mammalian species, species,

such as, for example, rodents and non-human primates. In some aspects, the conservative

variant of SEQ ID NOs: 6-23 may include similarly tagged as proteins of S proteins of other other mammalian mammalian

species, such as, for example, rodents and non-human primates (that is, the variations is/are

within the 140 amino acid sequence of the as protein). In S protein). In some some aspects, aspects, the the invention invention excludes excludes

SEQ ID NO: 5 as the monomeric as substrate. S substrate.

[0051] In some aspects, the monomeric as substrate comprises S substrate comprises aa recombinant recombinant Sas protein protein

comprising six additional histidine amino acids (i.e., a polyHis purification tag) on the C-

terminus of SEQ ID NO: 5, resulting in a molecular mass of 15,283 Da and being represented

by the sequence:

PCT/US2021/033016

[0052] SEQ ID NO: 6:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL

GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA HHHHHH Thus,

[0053] Thus, SEQNO: SEQ ID ID 6 NO: is6distinguishable is distinguishable fromfrom SEQNO: SEQ ID ID 5 NO: by5the by six the additional six additional

histidine amino acids on the C-terminus. The retention of the histidine tag may improve the

ability of the human monomeric aS substrate to S substrate to avoid avoid self-aggregation. self-aggregation. SEQ SEQ ID ID NO: NO: 66 is is

further distinguishable from, e.g., a conservative variant of SEQ ID NO: 5 (and SEQ ID NO:

6) wherein one or more amino acids are added to the N-terminus. In some aspects, a

conservative variant of SEQ ID NO: 5 wherein one or more amino acids are added to the N-

terminus is excluded. However, some aspects include N-terminus additions. Thus:

[0054]

[0054] SEQIDIDNO: SEQ NO: 7: 7:

HHHHHH MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0055] Additional purification tags are contemplated, including, e.g., FLAG, HA, Myc, and

V5, thus generating the following SEQ ID NOs:

[0056]

[0056] SEQIDIDNO: SEQ NO: 8: 8:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDD

[0057]

[0057] SEQIDIDNO: SEQ NO: 9: 9:

DYKDDDD MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

WO wo 2021/236678 PCT/US2021/033016

[0058]

[0058] SEQIDIDNO: SEQ NO: 10: 10:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDDK

[0059] SEQ ID NO: 11:

DYKDDDDK MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0060] SEQ ID NO: 12:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDK

[0061] SEQ ID NO: 13:

DYKDDDK MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0062]

[0062] SEQIDIDNO: SEQ NO: 14: 14:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YPYDVPDYA

[0063]

[0063] SEQIDIDNO: SEQ NO: 15: 15:

YPYDVPDYA MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0064]

[0064] SEQIDIDNO: SEQ NO: 16: 16:

WO wo 2021/236678 PCT/US2021/033016

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YAYDVPDYA

[0065]

[0065] SEQIDIDNO: SEQ NO: 17: 17:

YAYDVPDYA MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0066]

[0066] SEQIDIDNO: SEQ NO: 18: 18:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YDVPDYASL

[0067]

[0067] SEQIDIDNO: SEQ NO: 19: 19:

YDVPDYASL MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0068]

[0068] SEQIDIDNO: SEQ NO: 20: 20:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA EQKLISEEDL

[0069]

[0069] SEQIDIDNO: SEQ NO: 21: 21:

EQKLISEEDL MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0070]

[0070] SEQIDIDNO: SEQ NO: 22: 22:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA GKPIPNPLLGLDST

[0071]

[0071] SEQIDIDNO. SEQ NO. 23: 23:

GKPIPNPLLGLDST MDVFMKGLSK GKPIPNPLLGLDST MDVFMKGLSK AKEGVVAAAE AKEGVVAAAE KTKQGVAEAA KTKQGVAEAA GKTKEGVLYV GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0072] The The preparation preparationof of monomeric as substrate, monomeric polypeptide S substrate, fragments polypeptide thereof, fragments thereof,

mutants, truncations, derivatives, and splice variants that display substantially equivalent or

altered alteredasS activity activityrelative to the relative wild-type to the protein wild-type are likewise protein contemplated. are likewise These variants contemplated. These variants

may be deliberate, for example, such as modifications obtained through site-directed

mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are

producers of the a-synuclein protein. Included -synuclein protein. Included within within the the scope scope of of these these terms terms are are Sas proteins proteins

specifically recited herein, and all substantially homologous analogs and allelic variants

thereof.

[0073] Analogs may be made through conservative amino acid substitution. A A "conservative amino acid substitution" may include, for example, one in which the amino acid

residue is replaced with an amino acid residue having a similar side chain. Families of amino

acid residues having similar side chains include amino acids with basic side chains (e.g., lysine,

arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side

chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side

chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,

tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side

chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0074] A "non-essential" amino acid residue is a residue that can be altered from the wild-

type type sequence sequenceofof as Swithout abolishing without or, more abolishing or, preferably, without without more preferably, substantially altering aS's substantially altering S's

biological activity. Thus, a predicted non-essential amino acid residue in a as proteinis S protein is

preferably replaced with another amino acid residue from the same side chain family.

Alternatively, in another aspect, mutations can be introduced randomly along all or part of the

as coding sequence, S coding sequence, such such as as by by saturation saturation mutagenesis, mutagenesis, and and the the resultant resultant mutants mutants can can be be

screened for as biological activity S biological activity to to identify identify mutants mutants that that retain retain activity. activity. Following Following

mutagenesis of the nucleotide sequence for aS, the encoded S, the encoded protein protein can can be be expressed expressed

recombinantly, and the activity of the protein can be determined.

[0075] The monomeric as substrates produced S substrates produced herein herein exhibit exhibit aa decreased decreased tendency tendency to to self- self-

aggregate. In some aspects, the monomeric as substrate does S substrate does not not self-aggregate self-aggregate under under S- aS-

SAA conditions. In other aspects, the monomeric as substrate self-aggregates S substrate self-aggregates at at aa much much lower lower

level and/or at a much slower rate than the level or rate of self-aggregation of monomeric as S

substrate obtained by previous methods of preparing monomeric as substrate. In S substrate. In such such aspects, aspects,

the rate and/or level of self-aggregation can 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 monomeric

as substrate obtained S substrate obtained using using methods methods other other than than those those described described herein. herein. In In other other aspects, aspects, the the

monomeric aS substrate self-aggregates S substrate self-aggregates at at aa slower slower rate rate or or lower lower level level than than the the rate rate or or level level of of

aggregation of monomeric aS substrate with S substrate with soluble, soluble, misfolded misfolded Sas protein. protein. InIn such such examples, examples,

aS-SAA detection is S-SAA detection is still still successful successful because because aa synucleinopathy-positive synucleinopathy-positive sample sample may may be be

distinguished from mere self-aggregation of monomeric as substrateby S substrate bythe theintensity intensityof ofthe the

detection (relative fluorescence units) or the time period that fluorescence increase begins. The

decreased self-aggregation for monomeric as substrate described S substrate described herein herein can can be be aa result result of of differences in the method used to prepare the monomeric as substrate, including S substrate, including differences differences in the resulting monomeric as substrate composition. S substrate composition.

[0076] The method of preparing human monomeric as substrate or S substrate or aa conservative conservative variant variant

may comprise culturing a host cell comprising a nucleic acid sequence coding for human aS

protein or a conservative variant, the nucleic acid sequence comprising codons that have been

optimized to produce human as protein or S protein or aa conservative conservative variant. variant. Cells Cells may may be be cultured cultured in in

conventional nutrient media modified as appropriate for inducing promoters, selecting

transformants, or amplifying the genes encoding the desired sequences. The culturing media

may include buffers, nucleosides (such as adenosine and thymidine), antibiotics, trace

elements, and glucose or an equivalent energy source. Any other necessary supplements may

also be included at appropriate concentrations that would be known to those skilled in the art.

The culture conditions, such as temperature, pH, and the like, are those previously used with

the host cell selected for expression and will be apparent to the ordinarily skilled artisan. For

a discussion of strategies for producing recombinant proteins using E. Coli, see Gopal G.,

Kumar A., Protein J., 32(6):419-25 (2013), the disclosure of which is incorporated herein by

reference in its entirety.

[0077] The method of preparing monomeric as substrate may S substrate may include include any any of of the the

expression vectors described herein. In some aspects, the enterobacteria is E. Coli. In further

aspects, the nucleic acid sequence of the expression vector comprises a sequence having at least

95% identity with SEQ ID NO: 1. In yet further aspects, the expression vector is a plasmid

comprising a sequence having at least 95% identity with SEQ ID NO: 2. In additional aspects,

the codons have been optimized to avoid amino acid incorporation, including cysteine

misincorporation, in the expressed human aS protein or a conservative variant.

[0078] Once cells have been allowed to grow in culture for an appropriate amount of time

(e.g., 4-24 hours), the cells are lysed and monomeric as protein is S protein is purified purified from from the the lysed lysed cells. cells.

A variety of methods may be used to lyse cells, such as use of a French press, sonication,

freeze-thawing, chemical lysis, and microfluidizing. Purification may include a variety of

different purification steps, such as centrifugation, column purification, dialysis, and acid

precipitation to a pH of about 3.5 or less, optionally followed by addition of LPS and/or

ultrafiltration (10kDa to 300kDa).

[0079] In some aspects, the method of preparing monomeric aS substrate from S substrate from the the host host

cell comprises lysing the cells using a microfluidizer. Microfluidizers break cells with high

efficiency while maintaining intracellular content integrity by supplying constant, controlled

shear rates, resulting in large cell membrane fragments that facilitate subsequent protein

purification. Use of a microfluidizer for cell lysis can decrease the tendency of purified

monomeric as substrate to S substrate to self-aggregate. self-aggregate. An An example example of of aa suitable suitable microfluidizer microfluidizer is is the the

LM20 Microfluidizer Microfluidizer®High HighShear ShearFluid FluidHomogenizer Homogenizermanufactured manufacturedby byMicrofluidics. Microfluidics.

[0080] The The method method of of preparing preparing monomeric monomeric as substrate may S substrate may comprise comprise the the step step of of

separating the monomeric as substrate from S substrate from other other components components of of the the host host cell cell such such as as lipids. lipids.

In some aspects, obtaining the monomeric as substrate from S substrate from the the host host cell cell (e.g., (e.g., E. E. Coli) Coli)

comprises contacting the monomeric as substrate with S substrate with Lipid Lipid Removal Removal Agent Agent ("LRA") ("LRA") to to

remove lipid contaminants (i.e., cell components). Thus, the monomeric as substrate mixed S substrate mixed

with various other cell components is contacted with LRA after cell lysis, and the monomeric

as substrate is S substrate isremoved removedby by centrifugation, which which centrifugation, separates the protein separates from the lipids the protein (thelipids from the LRA (the LRA

and bound lipids go to the pellet fraction during centrifugation). Use of LRA for removal of

the undesired lipid components may improve the ability of recombinant monomeric aS S

substrate in the resulting composition to avoid self-aggregation. LRA is a commercially

available agent (available from Millipore Sigma) based on synthetic calcium silicate hydrate.

[0081] The The method methodfor forpreparing monomeric preparing as substrate monomeric or a or S substrate monomeric as substrate a monomeric S substrate

composition may comprise the step of initially separating the monomeric as substratefrom S substrate from lipids, such as LPS, or nucleic acids, such as DNA and RNA. In some aspects, obtaining the monomeric as substrate from S substrate from the the host host cell cell (e.g., (e.g., E. E. Coli) Coli) comprises comprises precipitation precipitation of of non- non- synuclein components by addition of hydrochloric acid (HCI) (HCl) to less than pH 3.50, including about pH 2.0 or lower. In some aspects, the method comprises adding LPS to the acid precipitated monomeric as substrate. S substrate.

[0082] In one aspect, obtaining the monomeric as substrate from S substrate from the the host host cell cell comprises comprises

precipitation of non-synuclein components by subjecting the human aS protein or S protein or aa

conservative variant to one or more acid precipitation steps at a pH of about 3.5 or less, e.g.,

first at pH 3.5 and again at pH 2, followed by chromatography, to yield the purified monomeric

aS substrate or a conservative variant. An example of such an aspect is depicted by the flow

chart shown in Figure 11.

[0083] Options to remove contaminants such as metal-binding proteins (e.g., Ferric Uptake

Regulatory Protein; FUR) from the monomeric as substrate composition S substrate composition include include iron-IMAC, iron-IMAC,

antibody depletion, genomic modification of endogenous E. coli FUR to include a purification

tag (other than histag or other purification tag used by the as protein), iron S protein), iron saturation saturation to to reduce reduce

binding to the nickel column, or elimination washes with Fe++ during nickel-IMAC nickel-IMAC.In Insome some

aspects, purification of the monomeric as substrate comprises S substrate comprises excluding excluding essentially essentially all all other other

proteins (e.g., metal-binding proteins). In further aspects, the metal-binding protein is a ferric

uptake regulator (FUR).

aS Protein Compositions S Protein Compositions

[0084] The isolated human monomeric as substrate or S substrate or conservative conservative variant variant composition composition

can also include a suitable medium for suspending and/or storing the protein. For example, in

some aspects, the monomeric as substrateor S substrate orconservative conservativevariant variantcomposition compositionincludes includesaa

buffer such as piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or phosphate buffered saline

(PBS). In further aspects, the composition consists essentially of one of SEQ ID NOs: 5-23

and PIPES. The monomeric as substrate may S substrate may be be substantially substantially free free of of other other materials materials such such as as

lipid contaminant from the lysed enterobacterial cell.

[0085] In some aspects, the human monomeric aS substrate or S substrate or conservative conservative variant variant

composition is purified SO so that it is essentially free of contaminants. Most of the contaminants

found found in inthe thepurified monomeric purified as substrate monomeric or conservative S substrate variant variant or conservative are proteins are of host cell proteins of host cell

origin, with only a few potential contaminants of human origin. MS/MS identifies proteins by

detecting fragments after enzymatic digestion and comparing the fragment footprint to the

footprints of known proteins, which are available in public databases. Thus, some fragments

may match microorganisms other than the host cell (e.g., E. coli). However, most prokaryotic

contaminating proteins will be from the host cell.

[0086] The resulting relative abundance of all human-originated proteins is extremely low

compared to monomeric as substrate.The S substrate. Themost mostrepresented representedcontaminants contaminantsof ofhuman humanorigin originare are

cytochrome B5 and keratin related peptides.

[0087] As for the contaminants with bacterial origins, most are from the enterobacterial

host cell (e.g., E. coli), which is the expression host. By far, the most likely and abundant

bacterial contaminant appears to be FUR from E. coli. This protein has affinity for iron and is

responsible for controlling iron's intracellular concentration. When iron is bound to FUR, the

iron becomes capable of binding DNA and can act as a regulator. Since iron and nickel are

both divalent cations, FUR's ability to bind iron is potentially the reason why FUR co-purifies

with monomeric as substrateduring S substrate duringIMAC IMACpurification. purification.S-SAA aS-SAA competent competent monomeric monomeric S as

substrate or conservative variant may contain lower concentration of FUR protein than self-

aggregating substrate. The potential mechanism by which FUR induces self-aggregation could

include ionic interaction between monomeric as substrate and S substrate and residual residual iron iron or or nickel nickel carried carried

WO wo 2021/236678 PCT/US2021/033016

by FUR. The molecular weight of FUR is practically the same as aS, which may hide the

contaminant in molecular weight based discriminating tools.

[0088] Some other notable contaminants from E. coli with relatively high abundance and

matching scores are catabolite gene activator, HIT-like protein, FKBP-type peptidyl-prolyl cis-

trans isomerase, and several ribosomal proteins. Catabolite gene activator is another DNA

binding protein like FUR, but it does not require a metal cofactor. HIT proteins are proteins

with histidine triad motifs that could be involved in binding zinc and have been shown to be

able to bind nucleotides. Therefore, the triad motifs might also have affinity for nickel, which

could make HIT proteins co-purify with monomeric as substrate. S substrate.

[0089] Accordingly, in some aspects, the human monomeric as substrate or S substrate or conservative conservative

variant composition is essentially free of other proteins. In some aspects, the other proteins

comprise metal-binding proteins, while in further aspects the metal-binding proteins comprise

FUR. 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.

[0090] Potential Potentialcontaminants contaminantsof of the the human monomeric human as substrate monomeric or conservative S substrate or conservative

variant composition, which are excluded from the composition in aspects of the invention, may

include: Ferric uptake regulation 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 resistance protein ArnA OS=Escherichia coli; 50S ribosomal

protein L27 OS=Escherichia coli; Formyltetrahydrofolate deformylase OS=Escherichia coli;

30S ribosomal protein S15 OS=Escherichia coli; Glucosamine--fructose-6-phosphate Glucosamine-fructose-6-phosphate

aminotransferase [isomerizing] OS=Escherichia coli; Regulator of sigma D OS=Escherichia

coli; Acyl-[acyl-carrier-protein]--UDP-N-acetylglucosamine Acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase wo WO 2021/236678 PCT/US2021/033016

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

radiotolerans; Ribosomal RNA large subunit methyltransferase A OS=Escherichia coli;

Nucleoside diphosphate kinase OS=Alcanivorax borkumensis; Uncharacterized HTH-type

transcriptional 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=Jannaschia 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 saccharolyticus; Spermidine N(1)-acetyltransferase OS=Escherichia OS=Caldicellulosiruptor

coli; 50S ribosomal protein L4 OS=Neisseria meningitidis serogroup C; Aminoglycoside 3'-

phosphotransferase OS=Escherichia coli; Putative uncharacterized 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 tengcongensis; Ribosomal RNA small subunit methyltransferase H OS=Thermoanaerobacter

OS=Clostridium acetobutylicum; Phosphoribosylformylglycinamidine cyclo-ligase

OS=Geobacillus sp.; Uncharacterized protein yhbW OS=Escherichia coli; Putative 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; Cobyrinic

WO wo 2021/236678 PCT/US2021/033016

acid A,C-diamide synthase OS=Rhodobacter capsulatus; ATP synthase subunit beta

OS=Rickettsia akari; UPF0371 protein M6_Spy1067 OS=Streptococcus pyogenes serotype;

NAD-reducing hydrogenase hoxS subunit alpha OS=Cupriavidus necator; Chaperone protein

DnaK OS=Escherichia coli; Urease subunit alpha OS=Yersinia enterocolitica serotype; 1-

deoxy-D-xylulose-5-phosphate synthase OS=Bordetella avium; and Translation initiation

factor IF-2 OS=Proteus vulgaris.

Methods Methodsfor forUsing Monomeric Using as Substrate Monomeric in SAA S Substrate in SAA

[0091] Human monomeric as substrate or S substrate or conservative conservative variant variant compositions compositions described described

herein having a decreased tendency to self-aggregate are useful as substrate proteins in aS- S-

SAA. Example aS-SAA methods include S-SAA methods include those those disclosed disclosed in in US20160077111A1 US20160077111A1 ("slow ("slow

assay"), US20210063416A1 ("fast assay"), and U.S. Nonprovisional Patent Application No.

17/154,966.

[0092] Examples have been included to explain more clearly particular aspects of the

invention.

EXAMPLES Example Example1:1:Synthesis Synthesisof of recombinant monomeric recombinant as substrate monomeric SEQ ID SEQ S substrate NO: ID 6 using NO: 6SEQ ID using SEQ ID

NO: NO: 22

[0093] E. coli BL21(DE3) was transformed according to the manufacturer's instructions

(Lucigen (Lucigen®E. E.cloni Express cloni® Chemically Express Competent Chemically Cells, Competent MA019 Cells, Rev. MA019 31OCT2016) Rev. with 31OCT2016) with

the expression vector comprising the plasmid represented by SEQ ID NO: 2. This plasmid

comprises a codon optimized nucleic acid sequence represented by SEQ ID NO: 1, which

encodes for the C-terminal HisTag as protein represented S protein represented by by SEQ SEQ ID ID NO: NO: 66 without without amino amino

acid misincorporation.

[0094] Bacterial pellets were grown in-house overnight using autoinduction media. Pellets

were tested for inclusion bodies using B-Per reagent and SDS-PAGE. as protein represented S protein represented by SEQ ID NO: 6 was highly expressed (~30% of all protein) and there were no inclusion bodies detected by B-Per. SEQ ID NO: 1 was verified by DNA sequencing.

Example 2: Purification of as protein by S protein by microfluidization microfluidization and and acid acid precipitation precipitation

[0095] The bacterial pellets containing aS protein were 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 min in lysis buffer (50mM NaH2PO4 pH: 8.0, 0.3M NaCl,

0.2mMEDTA, 0.2mM EDTA, 20mM 20mM Imidazole, Imidazole, 1mMPMSF, 0.0. 1mM PMSF, 1mM TCEP). 1mM The TCEP). cells The were cells resuspended were inin resuspended

a final volume of lysis buffer equivalent to 4X the weight of the pellet (80 mL lysis buffer to

20 g of pellet). The resuspended cells were degassed using a standard vacuum pump. The

resuspended cells were lysed by a microfluidizer (LM20 Microfluidizer. The Microfluidizer®). crude The lysate crude lysate

was clarified by centrifugation to remove large cellular debris.

[0096] The clarified lysate was titrated by stepwise addition of 1M HCI HCl during agitation.

After reaching a target pH, the acidified lysate was incubated with agitation for 20-60 min.

The acidified lysate was clarified by centrifugation, and the supernatant was neutralized using

1M NaOH to pH 8.00. The neutralized lysate was filtered by 0.22 um µm filters, and the material

was loaded onto a column with nickel-Sepharose resin.

[0097] Chromatography was carried out using standard protocols. After loading the

neutralized filtered lysate, the packed column was washed with a first wash buffer (50 mM

NaH2PO4 pH: NaHPO pH: 7.4, 7.4, 0.5M 0.5M NaCl, NaCl, 2020 mMmM Imidazole, Imidazole, 0.1 0.1 mMmM TCEP) TCEP) and and a a second second wash wash buffer buffer

(50 mM NaH2PO4 pH: NaHPO pH: 7.4, 7.4, 0.15M 0.15M NaCl, NaCl, 2020 mMmM Imidazole, Imidazole, 0.1 0.1 mMmM TCEP). TCEP). Protein Protein was was

eluted eluted with withelution buffer elution (50 (50 buffer mM NaH2PO4 pH: pH: mM NaHPO 7.4,7.4, 0.15M0.15M NaCl,NaCl, 250 mM250 Imidazole, 0.1 mM Imidazole, 0.1

mM TCEP), and elution fractions were collected on ice. The elution fractions were evaluated

by SDS-PAGE and Coomassie staining to determine the fractions with the lower amounts of

contaminants and higher amounts of recombinant aS protein.

PCT/US2021/033016

[0098] After After pooling poolingthethe elution fractions elution with high fractions with as and Slow high contaminants, and the pool was low contaminants, the pool was

dialyzed for 4-5 h in 1X PBS at a dilution factor of 1:200. A second dialysis was performed

(1:400) overnight for a total 1:80,000 dilution factor.

[0099] The dialyzed material was filtered using a 50,000 Dalton MWCO Amicon

centrifugal device. The 50kDa filtered material is the final as monomerproduct, S monomer product,and andprotein protein

concentration was determined by BCA, A280, or a combination of A280 and BCA for

increased accuracy. The protein was aliquoted into single use aliquots containing around 6.5

mg each. each.

[00100] Figures 2A and 2B show electrophoresis results for C-terminal HisTag as protein S protein

(SEQ ID NO: 6) prepared using the plasmid (SEQ ID NO: 2) comprising the nucleotide

sequence (SEQ ID NO: 1). The C-terminal HisTag as protein aliquot S protein aliquot was was separated separated into into two two

fractions. One fraction of the C-terminal HisTag as protein aliquot S protein aliquot was was contacted contacted with with DTT DTT

under disulfide bond reducing conditions. As shown in Figures 2A and 2B, the reduced and

non-reduced fractions, respectively, of the C-terminal HisTag as proteinwere S protein wereessentially essentially

identical when run at protein amounts per lane of 1 ug, µg, 2 ug, µg, and 4 ug. µg. No band corresponding

to dimer formation was visible. Accordingly, the plasmid (SEQ ID NO: 2) comprising the

nucleotide sequence (SEQ ID NO: 1) produces the cysteine misincorporation-free C-terminal

HisTag as proteinsequence S protein sequence(SEQ (SEQID IDNO: NO:6) 6)when whenexpressed expressedin inE. E.Coli. Coli.

Comparative Example 1: Gel electrophoresis of recombinant human monomeric as substrate S substrate

prepared without using optimized codons

[00101] Figure 3 shows electrophoresis results for C-terminal HisTag as protein (intending S protein (intending

SEQ ID NO: 6, but because of cysteine misincorporation, likely comprising a mix of SEQ ID

NO: 6 and SEQ ID NO: 6-Y136C) prepared using the plasmid (SEQ ID NO: 4) comprising the

nucleotide sequence (SEQ ID NO: 3) before and after treatment with DTT. Lane 1 (the first

lane from the left) shows various molecular weight fractions of an incubation mixture. Lane 2

WO wo 2021/236678 PCT/US2021/033016

shows the recombinant monomeric aS protein reagent (SD*). Lane 3 shows the filtrate (F)

through a 30 kDa cutoff filter. Lane 4 shows the retentate (R) caught by the 30 kDa cutoff

filter. Lane 5 shows the filtrate through a 50 kDa cutoff filter. Lane 6 shows the retentate

caught by the 50 kDa cutoff filter. The C-terminal HisTag as protein has S protein has aa nominal nominal molecular molecular

weight of ~15 kDa, but it runs slightly higher than the 17kDa marker in SDS-PAGE.

Accordingly, electrophoresis Accordingly, of the electrophoresis of filtrate throughthrough the filtrate the 30 kDa the cutoff 30 kDafilter in Lane cutoff 3 showed filter in Lane 3 showed

a band at ~15 kDa, corresponding to the C-terminal HisTag as protein. In S protein. In addition addition to to the the

intended 15 kDa recombinant monomeric folded aS protein, Figure 3, Lanes 4, 5, and 6

showed a band at ~36 kDa. After treatment of with DTT, the band at ~36 kDa disappeared,

leaving only the expected band at ~15 kDa, indicating the presence and separation of dimers

into monomers.

Example 3: aS-SAA of the S-SAA of the acid acid precipitated precipitated monomeric monomeric Sas substrate substrate

[00102] aS-SAA using the S-SAA using the acid acidprecipitated as monomer precipitated was was S monomer conducted by theby"fast conducted the "fast

assay" procedure, using the following parameters:

Parameter Fast Assay (FA)

Buffer ([] (mM) and pH) 100mM PIPES pH 6.50

[NaCl] (mM) 500

ThT (uM) (µM) 10

Recombinant C-terminal

histag histag aSyn Substrate (MW = 15,283mg/mmol) 15,283mg/mmol) (SEQ (SEQ IDID NO: 6)

[substrate] (uM) (µM) 19.6 uM µM

WO wo 2021/236678 PCT/US2021/033016

[substrate] (mg/mL) 0.3

Sample (uL) (µL) 40

Shaking type Orbital Orbital

Shaking speed (rpm) 600-800

Shaking time (min) 11

Incubation time (min) 29

Beads material Si3N4 SiN

Beads size (mm) 2.38

Amount of beads 1 (ea)

Temperature (°C) 37

Reaction volume (uL) (µL) 200 200

[00103] Figure

[00103] Figure 4A4Ashows shows S-SAA aS-SAA"fast "fast assay" assay" aggregation aggregationcurves (of(of curves three replicates three replicates

individually) in the presence of a confirmed PD sample, where the monomeric as substrate S substrate

was was purified purifiedbybyacid precipitation acid to pHto4.pH precipitation The4.monomeric as substrate The monomeric aggregated S substrate as aggregated as

expected in the presence of the PD sample, reaching Fmax between 60-70 hours. However,

the monomeric as substrate showed S substrate showed high high propensity propensity for for self-aggregation self-aggregation when when analyzing analyzing aa

CSF sample from a HC (Figure 4B).

[00104] Likewise, Figure 5A shows aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three

replicates individually) in the presence of a confirmed PD sample, where the monomeric as S

substrate was purified by acid precipitation to pH 3.5. The monomeric as substrate aggregated S substrate aggregated as expected in the presence of the PD sample, reaching Fmax between 60-70 hours, but showing greater variability than with the substrate produced using pH 4.0 acid precipitation.

The monomeric as substrate showed S substrate showed medium medium self-aggregation self-aggregation with with an an HC HC (Figure (Figure 5B). 5B).

[00105] Figure 6A shows aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three replicates replicates

was purified by acid precipitation to pH 3. At pH 3, the PD aggregation is as expected for only

two of the three wells. The third well showed a much lower fluorescence, which might be

explained by self-aggregation. More surprisingly, the HC (Figure 6B) was "reproducibly

positive," indicating that the substrate purified in this way is more prone to self-aggregation

(and further supporting the hypothesis that the third replicate from the PD sample could be self-

aggregation).

[00106] Figure 7A shows aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three replicates replicates

was purified by acid precipitation to pH 2.5. Strikingly, PD aggregation was heavily affected

in terms of poor reproducibility, delayed aggregation of the positive wells (75-90 hours), and

lack of lack of amplification amplificationforfor one one of the of replicates, which could the replicates, whichgenerate a false negative could generate a falseresult. negative result.

Conversely, the monomeric as substratedid S substrate didnot notself-aggregate self-aggregate(Figure (Figure7B). 7B).

Figure

[00107] Figure 8A and 8A and Figure Figure 8B show 8B show aS-SAA S-SAA "fast "fast assay" assay" aggregation aggregation curves curves (of three (of three

(Figure 8A) and in an HC (Figure 8B). The results were consistent with pH 2.5, with no self-

aggregation in the HC, but low PD aggregation, as only two of the three replicates were

positive.

WO wo 2021/236678 PCT/US2021/033016

Example 4: Addition of LPS to acid precipitated (pH 2.5) monomeric as substrateprior S substrate priorto to

aS-SAA of PD S-SAA of PD sample sample

[00108] LPS was added to the acid precipitated (pH 2.5) monomeric as substrate described S substrate described

in Example 3. Figure 9A shows aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three replicates replicates

individually) in the presence of a confirmed PD sample using 60,000 Endotoxin Units (EnU)

of LPS. The LPS accelerated the aggregation, which started around 50 hours. Two of the

replicates were clearly positive and showed reproducible aggregation, while the third one

displayed a much lower maximum fluorescence (Fmax). Despite the variability and low Fmax

of one replicate, this sample would be considered positive. 60,000 EnU of LPS induced very

high levels of self-aggregation (Figure 9B). An LPS addition of 600 EnU substantially

improved the reproducibility of the three replicates, which were all positive at the expected 50-

75 hours (Figure 9C). Strikingly, 600 EnU of LPD did not induce self-aggregation, allowing

clear identification of the HC sample as negative (Figure 9D).

Example 5: Addition of LPS to acid precipitated (pH [2.5) monomeric Sas 2.5) monomeric substrate substrate prior prior toto

aS-SAA of MSA S-SAA of MSA sample sample

[00109] LPS was added to the acid precipitated (pH 2.5) monomeric as substrate. Figure S substrate. Figure

10A and Figure 10B show aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of two two replicates replicates

individually) in the presence of a confirmed MSA sample using 600 EnU and 120 EnU,

respectively. The LPS induced aggregation with low fluorescence, consistent with MSA

diagnosis.

Example 5: Purification of as protein by S protein by microfluidization microfluidization and and double double acid acid precipitation precipitation

[00110] Figure 11 shows a flowchart of an example method for purifying monomeric aS S

substrate that, together with proper aS-SAA conditions, reduces, S-SAA conditions, reduces, slows, slows, or or prevents prevents altogether altogether misfolding and self-aggregation when used in an aS-SAA, yet retains S-SAA, yet retains its its activity activity in in the the presence of soluble, misfolded aS protein in a biological sample.

[00111] Thus, the bacterial pellets containing aS protein were 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 min in lysis buffer (50mM NaH2PO4 pH: 8.0,

0.3M NaCl, 0.2mM EDTA, 20mM Imidazole, 1mM PMSF, 1mM .1mMTCEP). TCEP).The Thecells cellswere were

resuspended in a final volume of lysis buffer equivalent to 4X the weight of the pellet (80 mL

lysis buffer to 20 g of pellet). The resuspended cells were degassed using a standard vacuum

pump. The resuspended cells were lysed by a microfluidizer (LM20 Microfluidizer made by

MicrofluidicsTM). MicrofluidiesTM). The crude lysate was clarified by centrifugation to remove large cellular

debris.

[00112] The clarified lysate was titrated by stepwise addition of 1M HCI HCl during agitation.

After reaching a target pH of 3.5, the acidified lysate was incubated with agitation for 20-60

min. min. The The acidified acidified lysate lysate was was clarified clarified by by centrifugation. centrifugation. The The clarified clarified lysate lysate was was again again titrated titrated

by stepwise addition of 1M HCI HCl during agitation. After reaching a target pH of 2.0, the

acidified lysate was incubated with agitation for 20-60 min. The double acidified lysate was

clarified by centrifugation, filtered by 0.45 um µm filters, and the supernatant was neutralized

using 1M NaOH to pH 8.00. The neutralized lysate was filtered by 0.22 um µm filters, and the

material was loaded onto a column with nickel-Sepharose resin.

[00113] Chromatography was carried out using standard protocols. After loading the

PCT/US2021/033016

contaminants and higher amounts of recombinant aS protein.

After

[00114] After poolingthe pooling theelution elution fractions fractionswith high with as and high lowlow S and contaminants, the pool contaminants, the was pool was

dialyzed for 4-5 h in 1X PBS at a dilution factor of 1:200 1:200.A Asecond seconddialysis dialysiswas wasperformed performed

(1:400) overnight for a total 1:80,000 dilution factor.

[00115] The dialyzed material was filtered using a 50,000 Dalton MWCO Amicon

centrifugal device. The 50kDa filtered material is the final as monomer product S monomer product and and protein protein

concentration was determined by BCA, A280, or a combination of A280 and BCA for

mg each.

[00116] Figures 12A-12D show aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three

replicates individually) of the resultant as proteinin S protein inthe thepresence presenceof ofthree threedifferent differentconfirmed confirmed

PD samples (Figures 12A-12C) and in an HC (Figure 12D). The PD samples showed

excellent reproducibility, and the HC showed no self-aggregation. This is surprising and

counter-intuitive in view of the self-aggregation exhibited in the HC using a single acid

precipitation step at pH 3.5 (Figure 5B) and the lack of desired aggregation in the PD sample

using a single acid precipitation step at pH 2.0 (Figure 8A).

Example 6: Purification of as protein by S protein by microfluidization, microfluidization, acid acid precipitation, precipitation, and and multiple multiple

filtration

[00117] The aS substratewas S substrate wasprepared preparedas asdescribed describedin inExample Example2, 2,with withthe theacid acid

precipitation pH target at about 3.1, except that one aliquot of the dialyzed, filtered aS substrate S substrate

was filtered a second time at 50kDa and one aliquot of the dialyzed, filtered as substrate was S substrate was

filtered a second time at 30kDa.

[00118] Figures 13A-13D show aS-SAA "fast assay" S-SAA "fast assay" aggregation aggregation curves curves (of (of three three

replicates individually) using the monomeric as substrate corresponding S substrate corresponding to to SEQ SEQ ID ID NO: NO: 66 expressed in E. coli strain BL21 (DE3) transformed using the plasmid represented by SEQ ID

NO: NO: 2, 2, purified purified by by acid acid precipitation precipitation to to pH pH about about 3.1, 3.1, and and further further purified purified by by aa second second filtration filtration

seeds (Figure 13C) and in an HC (Figure 13D).

[00119] Unless defined otherwise, all technical and scientific terms used herein have the

same meaning as commonly understood by one of ordinary skill in the art to which this

invention belongs.

[00120] Where a range of values is provided, each intervening value, to the tenth of the unit

of the lower limit unless the context clearly dictates otherwise, between the upper and lower

limit of that range and any other stated or intervening value in that stated range, is encompassed

within the invention. The upper and lower limits of these smaller ranges may independently

be included in the smaller ranges and are also encompassed within the invention, subject to any

specifically excluded limit in the stated range. Where the stated range includes one or both of

the limits, ranges excluding either or both of those included limits are also included in the

invention. invention.

[00121] The term "about" in conjunction with a number is intended to include 10% +10%of ofthe the

number. This is true whether "about" is modifying a stand-alone number or modifying a

number at either or both ends of a range of numbers. In other words, "about 10" means from

9 to 11. Likewise, "about 10 to about 20" contemplates 9 to 22 and 11 to 18. In the absence

of the term "about," the exact number is intended. In other words, "10" means 10.

[00122] The singular forms "a", "and", and "the" include plural referents unless the context

clearly dictates otherwise. Thus, for example, reference to "a sample" also includes a plurality

of such samples and reference to "a monomeric aS substrate" includes S substrate" includes reference reference to to one one or or more more

such molecule,and such molecule, and so SO forth. forth.

[00123] The complete disclosure of all patents, patent applications, and publications, and 30 Jul 2025

electronically available material cited herein are incorporated by reference, whether or not the specific

citation herein so states. The foregoing detailed description and examples have been given for clarity of

understanding only. No unnecessary limitations are to be understood therefrom. The invention is not

limited to the exact details shown and described, for variations obvious to one skilled in the art will be 2021273750

included within the invention defined by the claims.

[00124] In this specification, the terms “comprise”, “comprises”, “comprising” or similar terms

are intended to mean a non-exclusive inclusion, such that a system, method or apparatus that comprises a

list of elements does not include those elements solely, but may well include other elements not listed.

[00125] The reference to any prior art in this specification is not, and should not be taken as, an

acknowledgement or any form of suggestion that the prior art forms part of the common general

knowledge in Australia.

Claims

CLAIMS 01 Sep 2025

1. An expression vector for production of a protein comprising SEQ ID NO: 6, the expression

vector comprising:

a nucleic acid sequence coding for SEQ ID NO: 6 comprising codons that have been

optimized to produce SEQ ID NO: 6 when expressed by an enterobacterial host cell, 2021273750

wherein the nucleic acid sequence comprises a sequence having at least 95% identity with

SEQ ID NO: 1.

2. A method for preparing a protein comprising SEQ ID NO: 6, the method comprising:

transforming into an enterobacterial host cell an expression vector comprising a nucleic

acid sequence coding for SEQ ID NO: 6 comprising codons that have been optimized to

produce SEQ ID NO: 6 when expressed by the enterobacterial host cell, wherein the nucleic

acid sequence comprises a sequence having at least 95% identity with SEQ ID NO: 1;

culturing the enterobacterial host cell under conditions effective to produce the protein; and

obtaining the protein from the enterobacterial host cell.

3. The method of claim 2, wherein the enterobacteria comprises Escherichia coli.

4. A nucleic acid comprising a sequence having at least 95% identity with SEQ ID NO: 1,

wherein the nucleic acid sequence encodes for a protein of the sequence of SEQ ID NO: 6

when expressed in an enterobacterial host cell.

5. An expression vector comprising the nucleic acid sequence of claim 4.

6. A method for preparing a protein of the sequence of SEQ ID NO: 6, the method comprising:

introducing into an enterobacterial host cell a nucleic acid comprising a sequence comprising at

least 95% identity with SEQ ID NO: 1;

culturing the enterobacterial host cell under conditions effective to produce the protein; and obtaining the protein from the enterobacterial host cell. 01 Sep 2025

7. The method of claim 6, wherein the enterobacteria comprises Escherichia coli.

8. The method of claim 6, wherein the method further comprises an acid precipitation step to

purify the protein.

9. An enterobacterial host cell expressing the nucleic acid sequence of claim 4. 2021273750