ALPHA SYNUCLEIN SUBSTRATES AND METHODS FOR MAKING AND USING THE SAME
An expression vector is provided for production of human alpha-synuclein (αS) protein or a conservative variant thereof that exhibits a decreased tendency to self-aggregate in an αS seed amplification assay (SAA). The expression vector comprises a nucleic acid sequence coding for human αS protein or a conservative variant, the nucleic acid sequence comprising codons that have been optimized to produce human αS protein or a conservative variant when expressed by a host cell such as E. Coli . The codons have been optimized to avoid amino acid misincorporation in the expressed protein. Methods for purification of the expressed protein are also provided.
1 . A nucleic acid sequence having at least 95% identity with SEQ ID NO: 1, excluding SEQ ID NO: 3.
2 . An expression vector for production of a protein comprising SEQ ID NO: 6, the expression vector comprising a nucleic acid sequence comprising at least 95% identity with SEQ ID NO: 1, excluding SEQ ID NO: 3.
3 . The expression vector of claim 2 , further comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2, excluding SEQ ID NO: 4.
4 . A protein comprising SEQ ID NO: 6, prepared using the expression vector of claim 2 .
5 . 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 comprising at least 95% identity with SEQ ID NO: 1, excluding SEQ ID NO: 3;
culturing the enterobacterial host cell under conditions effective to produce the protein; and
obtaining the protein from the enterobacterial host cell.
6 . The method of claim 5 , wherein the expression vector further comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2, excluding SEQ ID NO: 4.
7 . The method of claim 5 , wherein the enterobacteria comprises Escherichia Coli.
8 . The method of claim 5 , wherein the obtaining comprises lysing the transformed cell using a microfluidizer.
9 . The method of claim 8 , wherein the lysing produces a cell lysate, and the method further comprises:
(i) clarifying the cell lysate; and
(ii) contacting the clarified lysate with a synthetic adsorbent of crystalline calcium silicate hydrate.
10 . The method of claim 8 , wherein the lysing produces a cell lysate, and the method further comprises:
(i) clarifying the cell lysate; and
(ii) titrating the clarified lysate with acid to a pH of about 3.5 or less.
11 . The method of claim 8 , wherein the lysing produces a cell lysate, and the method further comprises:
(i) clarifying the cell lysate;
(ii) titrating the clarified lysate with acid to a pH of about 2 or less; and
(iii) adding lipopolysaccharide.
12 . The method of claim 11 , further comprising, prior to adding the lipopolysaccharide:
(i) neutralizing the acidified lysate;
(ii) filtering the neutralized lysate;
(iii) subjecting the filtered lysate to chromatography to yield the protein;
(iv) filtering the protein;
(v) dialyzing the filtered protein; and
(vi) filtering the dialyzed protein.
13 . The method of claim 12 , further comprising filtering the dialyzed, filtered protein at least a second time.
14 . The method of claim 8 , wherein the lysing produces a cell lysate, and the method further comprises:
(i) clarifying the cell lysate;
(ii) titrating the clarified lysate with acid to a pH of about 3.5 to produce a first acidified lysate;
(iii) clarifying the first acidified lysate; and
(iv) acidifying the clarified, first acidified lysate to a pH of less than about 2 to produce a second acidified lysate.
15 . The method of claim 14 , further comprising:
(i) clarifying the second acidified lysate;
(ii) filtering the second acidified lysate;
(iii) neutralizing the filtered, second acidified lysate;
(iv) filtering the neutralized lysate;
(v) subjecting the filtered, neutralized lysate to chromatography to yield the protein;
(vi) filtering the protein;
(vii) dialyzing the filtered protein; and
(viii) filtering the dialyzed protein.