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 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.
2. An expression vector comprising the nucleic acid sequence of claim 1 .
3. The nucleic acid of claim 1 , wherein the nucleic acid sequence is incorporated into an expression vector to comprise a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2.
4. A composition comprising a protein consisting of SEQ ID NO: 6.
5. 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.
6. The method of claim 5 , wherein the nucleic acid sequence is incorporated into an expression vector to comprise a nucleic acid sequence having at least 95% identity with SEQ ID NO: 2.
7. The method of claim 5 , wherein the enterobacteria comprises Escherichia coli.
8. The method of claim 5 , 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 1 .
10. A modified enterobacterial host cell, the modified enterobacterial host cell expressing a heterologous nucleic acid that encodes for a protein of the sequence of SEQ ID NO: 6.
11. The modified enterobacterial host cell of claim 10 , wherein the enterobacteria comprises Escherichia coli.