Parkinson's disease (PD) is one of the common diseases in neurodegenerative diseases, so the early diagnosis of PD can effectively prevent the development process [1]. The pathophysiology of PD manifest as the loss of neurons in locus coeruleus and substantia nigra [2]. Studies have shown that PD pathogenesis is related to the accumulation of α-synuclein (α-syn) in substantia nigra, and the expression level of α-syn in cerebrospinal fluid can be used as an important specific biomarker for early diagnosis of PD [3-4]. Besides α-syn, Aβ42 show highly relevant in PD, because some PD patients exhibit amyloid plaques similar to those found in Alzheimer's diseases [5]. NF-L is a neuronal structural protein, which represents a key biomarker of nerve damage when its expression level elevating [5]. NF-L provides a quantitative indicator to track neurodegeneration [5]. Therefore, our project intends to obtain PD-specific markers NF-L, Aβ42, and α-syn by genetic engineering methods, and then verifies the accuracy of recombination proteins by ELISA assay. Finally, we expect to prepare for the acquisition of specific antibodies and the construction of early PD diagnosis kits.

1.1 Construction for pET-28a(+)-NF-L/Aβ-42/α-syn

We constructed pET-28a(+)-NF-L/Aβ-42/α-syn using Infusion recombination. First, we amplified NF-L, Aβ-42, and α-syn fragments by high-fidelity PCR, with a length of 1059 bp, 789 bp, and 522 bp, respectively. Fig. 1A showed correct bands with target size of three genes. Then, we used restriction endonuclease EcoRI and XhoI to linearize the pET-28a(+) vector backbone. Fig. 1B showed the pET-28a(+) vector. After agarose gel and bands recovery, we next used Infusion reaction to obtain recombinant plasmids.

After Infusion reaction assay, we directly the recombination plasmids into expression strain E.coli BL21. Fig. 2A showed the single colony on the plate. We then selected 3, 12, and 8 colonies for pET-28a(+)-NF-L, pET-28a(+)-Aβ-42, and pET-28a(+)-α-syn, respectively. Next, we used vector universal primers T7-Forward and T7-Terminal to detect target genes insertion by colony PCR. As showed in Fig. 2B, NF-L, Aβ-42, and α-syn gene fragments has been successfully inserted into the pET-28a(+) backbone.

Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Fig.3 showed the correct sequences of recombination plasmids. We chose correct colony for further IPTG induction.

1.2 Construction for pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry/α-syn-mCherry

In order to visualize the recombination proteins, we further constructed pET-28a(+)-NF-L-mCherry, pET-28a(+)-Aβ-42-mCherry, and pET-28a(+)-α-syn-mCherry plasmids by fusing mCherry fluorescent gene to the C-terminal of target genes. We constructed recombination plasmid by three fragments Infusion ligation, including target genes, mCherry gene, and linearized vector backbone. Thus, we designed PCR primer for amplification with adding appropriate overlap adaptor sequences. First, we amplified NF-L, Aβ-42, and α-syn fragments by high-fidelity PCR with specific adaptor primers, with a length of 1059 bp, 789 bp, and 522 bp, respectively. Fig. 4A showed correct bands with target size of three genes. Then, we amplified mCherry fluorescent gene by high-fidelity PCR with a length of 711 bp. Fig. 4B showed correct bands with target size of mCherry gene. The linearized pET-28a(+) vector backbone was the same as Fig. 1B.

After Infusion reaction assay, we directly the recombination plasmids into expression strain E.coli BL21. Fig. 5A showed the single colony on the plate. We then selected 3, 12, and 11 colonies for pET-28a(+)-NF-L-mCherry, pET-28a(+)-Aβ-42-mCherry, and pET-28a(+)-α-syn-mCherry, respectively. Next, we used vector universal primers T7-Forward and T7-Terminal to detect target genes insertion by colony PCR. As showed in Fig. 5B, NF-L, Aβ-42, α-syn , and mCherry gene fragments has been successfully inserted into the pET-28a(+) backbone.

Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Fig.6 showed the correct sequences of the fusion junction between target genes and mCherry fluorescent gene. We chose correct colony for further IPTG induction.

2.1 Protein expression

Based on the lac operator system in pET-28a(+) vector backbone, we used IPTG for protein prokaryotic expression. First, we used a final concentration of 1 mM/L IPTG for usual protein expression. Then, we set two temperature and different expression time for determining the optimal induction condition. The expected molecular weights of 6xHis-NF-L, 6xHis-NF-L-mCherry, 6xHis-Aβ-42, 6xHis-Aβ-42-mCherry, 6xHis-α-syn, and 6xHis-α-syn-mCherry are 44.8 kDa, 71.5 kDa, 34.0 kDa, 60.7 kDa, 23.3 kDa, and 50.0 kDa, respectively. As showed in Fig. 7, we can clearly observe protein bands of target genes. Interestingly, we found that the molecular weight of 6xHis-Aβ-42 and 6xHis-Aβ-42-mCherry are slightly larger than as predicted, suggesting that these two proteins might have some post-translational modifications. For 6xHis-NF-L and 6xHis-NF-L-mCherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level was highest after 6 hours of induction. For 6xHis-Aβ-42 and 6xHis-Aβ-42-mCherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level showed similar between after 3 hours and 6 hours induction, indicating that the protein synthesis rate had reached a plateau. Interestingly, 6xHis-α-syn showed different protein synthesis trends under 25°C and 37°C. Under 25°C, the protein expression level of 6xHis-α-syn was highest after 1 hours of induction, but as the induction time increased, the protein was further degraded. For 37°C, the protein expression level of 6xHis-α-syn was highest after 6 hours of induction. For 6xHis-α-syn-mCherry, the protein synthesis rate under 37°C is faster than that under 25°C, and the protein expression level was highest after 6 hours of induction.

We further used anti-His antibody for Western Blotting experiments and then quantified the induction amount of target proteins according to the relative gray value of the band. As showed in Fig. 8A, we found that the His antibody specificity is not particularly high, suggesting that we might need to optimize the relative concentrations of the primary and secondary antibodies, but we can still clearly see the bands of target proteins. Then, we used ImageJ software to calculate the relative gray value intensity of target proteins. The results were consistent with those of Coomassie Brilliant Blue staining in Fig. 7. Different temperatures have an effect on the correct folding and biological activity of proteins. Finally, except for 6xHis-α-syn, we chose 25°C and 37°C after 6 hours of induction for large-scale purification. For 6xHis-α-syn, we chose 25°C after 1 hours of induction and 37°C after 6 hours of induction for large-scale purification, respectively.

2.2 Protein His-tag purification

As showed in Fig. 9, we could clearly observe that the combination proteins with mCherry fluorescent tag have visible red precipitate. We further used His-tag purification kit to purify target proteins and then stained protein bands by Coomassie Brilliant Blue staining. As showed in Fig. 10 and Fig. 11, target proteins were enriched in elution components. Compared with proteins induced under 25°C, proteins induced under 37°C had better purification efficiency and less miscellaneous bands. In addition, we also found that the efficiency of protein binding with Ni-NTA beads is not particularly high, so we might need to optimize the conditions to obtain higher recovery yield. Then, we collected elution components for functional test.

3.1 Determination of protein fused with mCherry tag by fluorescence microscope

First, we determined the effectivity of target protein fused with mCherry tag by fluorescence microscope. After fixing sample and adding anti-fading agent, we detected mCherry signal using an excited light wavelength of 587nm and an emitted light wavelength of 610nm. As showed in Fig. 12, E.coli BL21 expressing pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry/α-syn-mCherry could be clearly observed with red fluorescence in cord-like structure.

3.2 Verification of target recombination protein by ELISA assay

We used ELISA assay to verify the accuracy of NF-L protein. As showed in Fig. 13A, the correlation between NF-L protein and absorbance 450nm is 0.9972, indicating that ELISA assay can accurately and effectively detect the content of NF-L in elution components. In elution components of protein expression under 25°C, the content of NF-L protein in E6 was the highest, reaching 25.31 ng/mL (Fig. 13B). Interestingly, in elution components of protein expression under 37°C, the content of NF-L protein in E2 was the highest, reaching 34.25 ng/mL (Fig. 13C).

Then, we used ELISA assay to verify the accuracy of Aβ-42 protein. As showed in Fig. 14A, the correlation between Aβ-42 protein and absorbance 450nm is 0.9983, indicating that ELISA assay can accurately and effectively detect the content of Aβ-42 in elution components. In elution components of protein expression under 25°C, the content of Aβ-42 protein in E6 was the highest, reaching 21.35 pg/mL (Fig. 14B). In elution components of protein expression under 37°C, the content of Aβ-42 protein in E6 was the highest, reaching 26.92 pg/mL (Fig. 14C).

Finally, we used ELISA assay to verify the accuracy of α-syn protein. As showed in Fig. 15A, the correlation between α-syn protein and absorbance 450nm is 0.994, indicating that ELISA assay can accurately and effectively detect the content of α-syn in elution components. In elution components of protein expression under 25°C, the content of α-syn protein in E6 was the highest, reaching 97.54 ng/mL (Fig. 15B). In elution components of protein expression under 37°C, the content of α-syn protein in E6 was the highest, reaching 98.32 ng/mL (Fig. 15C).

Our experiment did not optimize the protein expression conditions, and fermentation regulation plays a crucial role in yield. We can continue to optimize the expression conditions of the strains, such as oxygen levels, inoculum size, and pH, to improve production and to achieve the amount of protein required for antigen preparation.

In our expression process, we added inducers IPTG. We can explore more environmentally friendly production methods by modifying promoter of vector backbone or replacing the inducers with external regulatory conditions, such as temperature and light exposure, to induce expression through these greener and harmless approaches.

Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci. (2001) 2:492–501.

Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. Cell Tissue Res. (2004) 318(1):121-34.

Giasson BI, Murray IV, Trojanowski JQ, Lee VM, A. hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem. (2001) 276:2380–6.

Hoyer W, Cherny D, Subramaniam V, Jovin TM. Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro. Biochemistry. (2004) 43:16233–42.

Xiang C, Cong S, Tan X, Ma S, Liu Y, Wang H, Cong S. A meta-analysis of the diagnostic utility of biomarkers in cerebrospinal fluid in Parkinson's disease. NPJ Parkinsons Dis. (2022) 29;8(1):165.