Alpha-Synuclein, Recombinant, Human

Cosmo Bio

Catalog No.: CSR-SYN04-2

Product Description

E. coli expressed human α-synuclein has been widely used in structural and functional studies. Masuda et. al. (1)  have reported that approximately 20% of E. coli expressed human α-syn is mistranslated resulting in a Cys residue instead of Tyr at position 136 and causing an increase in the levels of dimeric α-synuclein.

CSR-SYN04 is E. coli human alpha-Synuclein (human) expressed from a plasmid in which the TAC codon coding for Tyr136 in the human protein is replaced by TAT to produce protein expressing Tyr at position 136 homogenously. It can be used as human α-syn under similar conditions to that of in vivo.

This product can also be used for comparison (control) of α-Synuclein Fibrils (CSR-SYN03).

Source: Human
Expression System: E.coli
Concentration: 1mg/mL
Purity: ≥ 90% (SDS-Page)
Storage: -20°C

References

(1) Masuda M, Dohmae N, Nonaka T, Oikawa T, Hisanaga S, Goedert M, Hasegawa M. Cysteine misincorporation in bacterially expressed human alpha-synuclein. FEBS Lett. 2006 Mar 20;580(7):1775-9. doi: 10.1016/j.febslet.2006.02.032. Epub 2006 Feb 24. PMID: 16513114.

Related Products

Product	Cat. No.
Alpha-Synuclein Aggregation Assay Kit	CSR-SYN01
Amyloid Fluorescent Staining Kit	CSR-SYN02
Alpha-Synuclein Fibrils, Human	CSR-SYN03
Alpha-Synuclein, Human	CSR-SYN04
Alpha-Synuclein Fibrils, Mouse	CSR-SYN05
Alpha-Synuclein, Recombinant, Mouse	CSR-SYN06

CSR-SYN03 / CSR-SYN04 FAQ

Q1. How is CSR-SYN04 prepared?
Full-length α-synuclein is expressed in E. coli. Monomers purified from the lysate are subjected to ultracentrifugation (160,000 × g, 20 min), and the resulting supernatant is designated as CSR-SYN04.

Q2. What are the molecular weights of CSR-SYN03 and CSR-SYN04?
The molecular weight of CSR-SYN04 is 14.5 kDa. The molecular weight of CSR-SYN03 cannot be determined because it contains multimers and polymers.

Q3. Regarding codon optimization in CSR-SYN04, what changes were made to the construct sequence?
The full-length α-synuclein sequence was modified only at residue Tyr136, where the codon was changed from TAC to TAT. No other changes were introduced.

Sequence:
ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTATGAACCTGAAGCCTAA

Reference: https://pubmed.ncbi.nlm.nih.gov/16513114/

Q4. What are the amino acid sequences of CSR-SYN03 and CSR-SYN04?
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

Q5. Does CSR-SYN04 have any tags?
No, CSR-SYN04 does not contain any tags.

Q6. Can large quantities be purchased from a single lot?
For CSR-SYN03, up to 40 vials (equivalent to 4 mg) can be manufactured and supplied from the same lot. Please inquire for pricing details.

Q7. Can CSR-SYN03 be detected using a phospho-specific antibody?
No. CSR-SYN03 is a fibrillized recombinant α-synuclein monomer purified from E. coli and is not phosphorylated. Therefore, it cannot be detected by phospho-specific antibodies. Any observed signal on a Western blot is considered to be due to non-specific cross-reactivity.

Q8. How is CSR-SYN03 prepared?
1. Fibrillize CSR-SYN04 at 37 °C.
2. Ultracentrifuge and collect the fibrils as a precipitate (non-fibrillized monomers remain in the supernatant and are removed).
3. Add an appropriate amount of physiological saline to the precipitate and ultracentrifuge again to further remove any remaining monomers.
4. Resuspend the fibrils in physiological saline. The sample subjected to ultrasonic treatment is designated as CSR-SYN03.

Q9. Is there evidence that autoclaving at 134 °C for 20 min or at 121 °C for 20 min in the presence of ≥3% SDS eliminates seeding (aggregation) activity?
Residual seeding activity has been observed after autoclaving at 121 °C for 20 minutes. Therefore, we recommend following the inactivation conditions specified in the data sheet.

Q10. Are there data showing that seeding (aggregation) activity is eliminated by autoclaving (134 °C, 20 min) or by autoclaving with SDS (≥3%) added (121 °C, 20 min)?
Please refer to the following publication: https://pubmed.ncbi.nlm.nih.gov/29669601/

Q11. Can CSR-SYN03 be detected using a phospho-specific antibody?
It has been confirmed that the supernatant obtained after fibrillation during CSR-SYN03 production contains almost no monomers. Considering the two subsequent ultracentrifugation steps, any residual monomers are presumed to be almost completely removed.

Q12. Does CSR-SYN03 contain monomers?
The supernatant after the fibrillation step contains almost no monomer. Given the additional two ultracentrifugation steps, any remaining monomers are believed to be nearly completely eliminated.

Q13. When CSR-SYN03 is dissolved in SDS sample buffer and analyzed by Western blotting, a band corresponding to the monomer molecular weight is observed. Does this indicate the presence of monomers?
When CSR-SYN03 is dissolved in SDS sample buffer and analyzed by Western blotting, a band at the monomer molecular weight (14.5 kDa) may appear. This does not necessarily indicate the presence of monomers in the sample. It is unlikely that monomers remain after purification or are generated during sonication. The observed band most likely results from a small amount of monomer released during SDS treatment.

Q14. Is information available on the endotoxin level of the product?
Measurements were conducted by a collaborating research group at the Tokyo Metropolitan Institute of Medical Science, which developed the production technology.
Reference: https://www.nature.com/articles/srep30891
As an example, the endotoxin level was reported to be 10.6–11.3 endotoxin units per milligram of protein.

Q15. How is the seeding activity of CSR-SYN03 evaluated?
Seeding activity is evaluated by adding 1 µg of CSR-SYN03 to 100 µL of CSR-SYN04 and monitoring Thioflavin T fluorescence over time. Seeding activity is confirmed when the fluorescence intensity exceeds the threshold after 15–20 hours of incubation.