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. 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, wherein the nucleic acid sequence comprises a sequence having at least 95% identity with SEQ ID NO: 1 wherein the protein has alpha-synuclein activity.
2. The expression vector of claim 1 , further comprising a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2.
3. 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.
4. The method of claim 3 , wherein the expression vector further comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2.
5. The method of claim 3 , wherein the enterobacteria comprises Escherichia coli.
6. The method of claim 3 , wherein the obtaining comprises lysing the transformed cell using a microfluidizer.
7. The method of claim 6 , 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.
8. The method of claim 6 , 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.
9. The method of claim 6 , 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.
10. The method of claim 9 , 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.
11. The method of claim 10 , further comprising filtering the dialyzed, filtered protein at least a second time.
12. The method of claim 6 , 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.
13. The method of claim 12 , 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.