R E S U L T S
Module I: Aptamer Binding
Qualitative analysis: Electrophoretic mobility shift assay (EMSA)
An electrophoretic mobility shift assay (EMSA) is a common affinity electrophoresis technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence. In the present study, EMSA was employed for affinity test of the aptamers.
Figure 1: Principle of Electrophoretic Mobility Shift Assay (EMSA). In this assay, aptamers are incubated with thrombin or left unbound, followed by non-denaturing polyacrylamide gel electrophoresis to separate the components of the system. The appearance of free bands indicates aptamers that do not bind to thrombin, whereas shifted bands reveal aptamers that successfully bind to thrombin.
After thrombin and aptamers were diluted with proper buffer, reaction systems were built with a gradient of aptamers. 15-mer, 29-mer and 40-mer aptamers were tested, and a gradient of concentration of thrombin were applied to reflect the binding affinity. After the aptamers were co-incubated with thrombin for 60 min, an 12% non-denaturing polyacrylamide gel electrophoresis was performed. The gel was then stained by fluorescent dye. GelRed was used as the DNA dye. Random extension was added to aptamer, in order to enhance GelRed incorporation.
Figure 2: The design of aptamer-linker-probe complex. This structure enlarged the DNA molecule while its binding activity was not weakened.
The electrophoresis showed that the aptamers showed rather strong binding affinity (Figure 1-2). The shift bands became more clear as the concentration of thrombin increased. 15-mer and 29-mer aptamers had a clear shift band and a clear non-shift band. 40-mer aptamer was suspected to form multimers, causing a strong band at the sampling hole and unclear bands at the target sites.
Figure 3: Native-PAGE results of EMSA. In the two figures, lane 1, marker; lane 2, control group with 15-mer, 29-mer and 40-mer aptamers and NO thrombin. All aptamers were at a concentration of 10 pM. Figure 3A: lane 3-5, 0.45 pM thrombin incubated with respectively 29-mer+40-mer, 15-mer+40-mer, 15-mer+29-mer; lane 6-8, 15-mer aptamer incubated with gradient thrombin content of 0.45, 0.9 and 1.8 pmol. Figure 3B: lane 3-5, 29-mer aptamer incubated with gradient thrombin content of 0.45, 0.9 and 1.8 pmol.; lane 6-8, 40-mer aptamer incubated with gradient thrombin content of 0.45, 0.9 and 1.8 pmol.
Quantitative analysis: Surface Plasmon Resonance (SPR)
EMSA provided a relatively rudimentary validation of the binding interactions. To achieve a more quantitative and precise characterization of the interactions between the 29-mer/40-mer aptamers and thrombin, Surface Plasmon Resonance (SPR) was employed for testing.
Figure 4: Schematic of the Surface Plasmon Resonance (SPR) Experiment. Biotinylated aptamers are immobilized on the SPR chip via streptavidin (SA) binding. Thrombin is then introduced to flow over the chip. When binding occurs, it causes a change in the resonance angle, indicating an interaction between the thrombin and the aptamers.
Briefly, streptavidin (SA) was amino-conjugated to capture biotinylated aptamers and seal the chip with bovine serum albumin (BSA) to prevent non-specific binding of thrombin. Then gradient diluted thrombin was loaded to obtain the corresponding curves within SPR buffer. Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD). Detailed operational procedures can be found on the Experiments-Surface Plasmon Resonance (SPR) page.
Figure 5: Surface Plasmon Resonance (SPR) results of aptamer-thrombin binding. The sensorgrams illustrate the binding interactions between the 29-mer/40-mer aptamers and thrombin, showing real-time changes in refractive index. Curves represent the association and dissociation phases, providing insights into the binding kinetics and affinity of the aptamers for thrombin. A & C: Thrombin was subjected to binding and dissociation tests by flowing gradient-diluted samples over the chip. B & D: Partial experimental results were fitted with 1:1 binding kinetic model in order to calculate dissociation constant (KD).
| Aptamer | General Kinetics model | Quality Kinetics Chi² (RU²) | 1:1 binding ka (1/Ms) | kd (1/s) | KD (M) | tc |
|---|---|---|---|---|---|---|
| 29 | 1:1 binding | 1.03e0 | 2.14e+5 | 1.74e-4 | 8.16e-10 | 4.81e+7 |
| 40 | 1:1 binding | 4.58e+1 | 1.59e+5 | 1.85e-1 | 1.17e-6 | 5.29e+7 |
Table 1: Parameters fitted from 1:1 binding kinetic model. RU: resonance units; ka: association rate constant (M-1s-1); kd: dissociation rate constant (s-1); KD: equilibrium dissociation constant (M); tc: flow rate-independent component of the mass transfer constant.
As shown above, aptamer 29 demonstrated a strong affinity for thrombin, with a dissociation constant (KD) of 0.816 nM, while aptamer 40 had a much lower affinity, with a KD of 1170 nM. Due to the high affinity between aptamer 29 and thrombin, the dissociation was incomplete when thrombin concentrations were high, this could be improved by increasing the flow rate during the experiment. A 1:1 binding kinetic model was used based on the assumption that each aptamer binds to a single site on thrombin. Surface Plasmon Resonance (SPR) experiments meticulously verified the binding interactions between aptamers and thrombin. By accurately determining the association and dissociation constants, we have significantly bolstered our confidence in the results obtained from our Electrophoretic Mobility Shift Assays (EMSA), laying a solid groundwork for the foundational concepts necessary for our subsequent system development.
Module II: Protease-based Circuit
Protein Solubility Analysis
Since our attempt of purifying PPVp, FKBP-cPPVp, FRB-nPPVp, and G-PPV-Y(the substrate of PPVp) without soluble tags ended up in failure, we further examined the solubility of our target proteins. It is achieved by 3s ultrasonication on ice + 10s interval, power 300W, for 40 minutes to completely destruct bacterial structure. Then the sample is centrifuged. The soluble and insoluble components will appear in the supernatant and the precipitate respectively. With 5×SDS loading buffer treated, the two parts can be used for downstream SDS-PAGE analysis. As a control, EGFP, which is soluble in E.coli, is also expressed and analyzed with the same protocol.
Solubility analysis of PPV and its split protease
- On-column refolding method
Figure 6: Solubility analysis of PPV through SDS-PAGE, its split form and substrate. SU: supernatant after ultrasonication; PU: precipitate after ultrasonication. Figure 6A: Solubility analysis of FKBP-cPPV and FRB-nPPV. Figure 6B: Solubility analysis of PPVp and G-PPVs-Y(GFP-PPV cut site-YFP). MW: FRB-nPPV: 24.9kDa; FKBP-cPPV: 27.3kDa; PPVp: 27.7kDa; G-PPVs-Y: 22.3kDa.
The result shows that almost all of the FRB-nPPV and FKBP-cPPV exist in PU but not SU, which indicates the insoluble state(inclusion body) of the proteins. In contrast, the EGFP control mainly appears in the soluble component(SU), proving the correctness of our protocol. Figure 6B is similar to figure 6A. Both of the proteins are insoluble. The unlabeled electrophoresis bands are due to sample loading mistakes.
- Solubility tag method
Figure 7: Effects of Soluble tags. When total proteins are extracted (AU) and centrifuged, the solubility of the proteins can be assessed. Soluble tags are expected to enhance the solubility of the target proteins.
Figure 8: Solubility analysis of PPV, its split form and substrate, all with MBP tag, through SDS-PAGE. AU: after ultrasonication; SU: supernatant after ultrasonication; PW: precipitate after washing.
The result showed that All MBP tagged proteins are soluble, for there are bands in all AU, SU, PW.
Solubility analysis of TEV
- On-column refolding method
Figure 9: Protein solubility analysis of TEV through SDS-PAGE. AU: after ultrasonication; SU: supernatant after ultrasonication; FS: final supernatant. Lane1, AU 1-1, i.e., total proteins of bacteria after ultrasonication; Lane2, SU1-1, i.e., proteins in the supernatant after ultrasonication; Lane3, FS1-1, i.e., proteins in the final supernatant. MW of TEV is around 31.0 KDa.
This result demonstrated that bacteria expressed TEV successfully. TEV is insoluble as there is no corresponding band in supernatant after ultrasonication. Purification of TEV in inclusion bodies proved to be successful, for about half of TEV appears in the final supernatant.
Protein Purification
Because our system is an in vitro detection system, it’s essential for us to express proteins and then purify them. We have chosen three strategies to express proteins. Directly expression with 6xHis tag but without solubility tags, expression with both 6xHis tag and solubility tags and expression with cell-free system. Methodology of ours for purification is affinity chromatography. To be specific, we use nickel affinity chromatography to purify proteins with 6xhis tag (with or without solubility tags), and use glutathione affinity chromatography to purify proteins expressed by cell-free system. For nickel affinity chromatography, we use both ÄKTA system and gravity chromatography. For glutathione affinity chromatography, we use ÄKTA system.
Figure 10: Overview of protein purification work flow. The protein purification process begins with transformation (1), where the desired gene is introduced into a host cell. This is followed by selection (2) of successfully transformed cells on a selective medium. Next, protein production (3) is induced in the selected cells, which are then subjected to cell lysis (4) via ultrasonication to release the proteins. The lysate is then subjected to protein purification (5) using an AKTA system or gravity column. The purified proteins are analyzed (6) to assess the success of the purification process, and finally, an activity assay (7) is performed to evaluate the functionality of the purified proteins.
Furthermore, for proteins that are not expressed with solubility tags or cell-free system, they usually form inclusion bodies and we need new strategy to tackle this tricky problem. On-column refolding is the very solution applied by us.
Figure 11: Workflow for extracting and purifying target proteins from inclusion bodies. AU: after ultrasonication (total proteins). SU: supernatant after ultrasonication. PU: precipitate after ultrasonication. SW: supernatant after washing. PW: precipitate after washing. PD: precipitate after dissolution. FS: final supernatant.
Finally, to test whether the affinity chromatography works as expected, we have done SDS-PAGE analysis to see the purification results.
Purification of PPV and its split protease
- On-column refolding method
Figure 12: Elution profile of the nickel affinity chromatography of cPPV, nPPV, PPV, G-PPVcutsite-Y without solubility tags. Method used for dissolving those proteins from inclusion bodies is on-column refolding. Blue line stands for UV curve and Red line stands for conductivity curve. ****Figure 12A, elution profile of cPPV. Peak A stands for transmission peak, peak B stands for elution peak of cPPV. Figure 12B, elution profile of nPPV. Peak A stands for transmission peak, peak B stands for elution peak of nPPV. Figure 12C, elution profile of PPV. Peak A stands for transmission peak, peak B stands for elution peak of PPV. Figure 12D, elution profile of G-PPVcutsite-Y. Peak A stands for transmission peak, peak B stands for elution peak of G-PPVcutsite-Y.
Figure 13: SDS-PAGE analysis of the cell lysate, the transmission peak and the elution peak after nickel affinity chromatography through AKTA or gravity chromatography. Those proteins were expressed without solubility tags. Figure 13A, Lane2-4, total proteins of bacteria expressing cPPV, elution peak, transmission peak of cPPV; Lane5-7, total proteins of bacteria expressing nPPV; Lane9-10, elution peak and transmission peak of nPPV. All the proteins are purified through AKTA. Figure 13B, Lane2, total proteins of bacteria expressing G-PPVcutsite-Y; Lane3, elution peak of G-PPVcutsite-Y through AKTA; Lane4, total proteins of bacteria expressing PPV; Lane5, elution peak of PPV through AKTA; Lane6, elution peak of G-PPVcutsite-Y through gravity chromatography; Lane7, elution peak of PPV through gravity chromatography. PPV wasn’t purified successfully in this picture. Figure 13C, Lane2-3, elution peak of PPV through gravity chromatography; Lane4-5, elution peak of PPV through AKTA after ultrafiltration inside the ultrafiltration membrane; Lane6-7, elution peak of PPV through AKTA after ultrafiltration outside the ultrafiltration membrane. Lane4-5&Lane6-7 showed that the ultrafiltration membrane was integrated and leak of proteins did not happen.
As shown above, we successfully purified nPPV, cPPV, PPV and their substrate G-PPVcutsite-Y without solubility tags.
- Solubility tag method
Figure 14:SDS-PAGE analysis of the elution peak of proteins expressed by bacteria through gravity chromatography. Lane1, elution peak of proteins containing MBP-PPV; Lane2, elution peak of proteins containing MBP-nPPV; Lane3, elution peak of proteins containing MBP-cPPV; Lane4, elution peak of proteins containing MBP-G-PPV-Y.
The results showed that we had successfully purified MBP-cPPV and MBP-G-PPV-Y, however, we failed to purify MBP-PPV and MBP-nPPV.
- Cell-free system method
Figure 15: SDS-PAGE analysis of proteins expressed by the ALiCE ® Cell-Free Protein Expression System. Figure 3A, SDS-PAGE analysis of positive control EYFP expressed by cell-free system. Lane2, negative control (ALiCE reaction mixture only) ; Lane3, reaction mixture with EYFP expression; Lane4, reaction mixture diluted two times with EYFP expression; Lane5, reaction mixture diluted twenty times with EYFP expression.
We proved that this cell-free protein expression system was feasible.
Purification of TEV
- On-column refolding method
Figure 16: SDS-PAGE analysis of the elution peak after gravity chromatography. Lane1-3, elution peak after gravity chromatography.
The results demonstrated that TEV was successfully purified.
Protease Activity Verification
Since our system relies on the protease both amplifying the signal and triggering the release of the final colloidal gold output, it is crucial to verify the target protease activity to ensure that the enzymes used in our experiments are active and functioning as expected. To achieve this, we designed a experiment to verify the enzyme activity under controlled conditions (you can find more detailed information about this experiment in our protocol). We validated the activity of two intact proteases and one split protease. For the intact TEV and PPV proteases, we mixed a calculated amount of the enzyme with its corresponding substrate and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were taken at different time points. The reaction was stopped with SDS loading buffer, followed by electrophoresis. Enzyme activity was confirmed by observing the reduction in substrate and the presence of cleavage product bands. For the split PPV protease, we additionally added a fixed amount of rapamycin to induce protease dimerization and activation.
Figure 17: Principle of protein activity assay. Substrates are incubated with proteases (PPV/TEV). Samples are extracted at different time points and subjected to denaturing polyacrylamide gel electrophoresis. The largest mass band represents the uncut substrate, while the two smaller mass bands indicate the cleaved products.
Figure 18: Enzymatic activity assay of TEV protease extracted from inclusion bodies.
MW:MBP-tevS-G-Y:65.6kDa G-Y:22.6kDa MBP:43.1kDa Reaction is stopped with 5×SDS loading buffer at different time points.
The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate. The top band corresponds to the intact substrate (MBP-tevS-G-Y), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (MBP and G-Y), which increase over time. Samples were taken at 0, 10, 30, 60, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes. The results confirm that the TEV protease extracted from inclusion bodies is active.
Figure 19: SDS-PAGE analysis of protease activity verification in split and full-length PPV constructs. Figure 19A: Lane M: Marker; Lane 0: Split-1 at 0 min; Lane Split-1: Split PPVp with 350 nM rapamycin at 10, 30, and 120 min; Lane Split-N: Split PPVp without rapamycin at 10, 30, and 120 min; Lane cPPV: Full-length FKBP-cPPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane nPPV: Full-length FRB-nPPVp with 1400 nM rapamycin at 10, 30, and 120 min. Figure 19B: Lane M: Marker; Lane Split-2: Split PPVp with 700 nM rapamycin at 10, 30, and 120 min; Lane Split-3: Split PPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane PPV: Full-length PPVp with its substrate at 10, 30, and 120 min.
The results show that both the full-length PPVp and the split PPVp (when induced by rapamycin) exhibit protease activity, as evidenced by the reduction in substrate over time. In contrast, a single split PPVp cannot cleave the substrate. However, when two split PPVs are combined without rapamycin induction, there is minimal cleavage activity, as indicated by the slight reduction in substrate from 10 to 30 and 120 minutes. The amount of substrate reduction is significantly less than that observed with rapamycin induction, leading to the conclusion that rapamycin is necessary for the optimal activity of the split PPVp.
Module III: Crosslinking between Aptamer and Protease
Click chemistry based crosslinking
To construct a molecular machine featuring a highly efficient sensor, such as an aptamer, and an upstream signal transporter, like a split protease, we have devised crosslinking via click chemistry to do this task firstly.
Figure 20: Concept diagram of click chemistry-based crosslinking between an aptamer and split protease. The aptamer is modified with dibenzoazacyclooctyne (DBCO), while the split protease contains p-Azido phenylalanine (p-AzF). These components covalently bond when incubated at room temperature, creating a stable complex that recognize biomarkers.
Through Gibson assembly, we successfully constructed the pET28a_nPPVp/cPPVp plasmids, which were subsequently verified by sequencing. Following site-directed mutagenesis, sequencing was again conducted to confirm the introduction of the desired mutations.
Figure 21: Sequencing verification of plasmids. (A-B) Sequencing results for the pET28a_nPPVp plasmid, showing the N-terminal and C-terminal regions, respectively. (C) Sequencing verification of the pET28a_nPPVp_mut, with red boxes highlighting the mutations. (D-E) Sequencing results for the pET28a_cPPVp plasmid, detailing the N-terminal and C-terminal regions, respectively. (F) Sequencing verification of the pET28a_cPPVp_mut, with red boxes indicating the mutations.
We induced protein expression in co-transformed bacteria when the OD600 reached approximately 0.8 by adding IPTG and L-arabinose, along with p-AzF. SDS analysis of total proteins in the cell lysates before ultrasonication was conducted to verify the expression of the target proteins. The expression of the proteins was confirmed; however, we encountered challenges in purifying the proteins, which may have been due to low protein yield.
Figure 22: SDS analysis of total proteins in cell lysates before ultrasonication. Lane 2-3: Total proteins from cell lysates of bacteria expressing hCG. Lane 4-5: Total proteins from cell lysates of bacteria expressing nPPV (15.2kDa) and cPPV (16.8 kDa), respectively.
Thus, we chose to use both AU (after ultrasonication) and SU (supernatant after ultrasonication) components for the protein ligation reaction. Despite employing very gentle warming and cooling during the process, the DBCO-modified aptamer consistently exhibited large molecular weight multimerization bands. Notably, no new bands appeared in the reaction system where the protein was added, indicating that the ligation was not successful.
Figure 23: Protein ligation reaction utilizing click chemistry. 29: DBCO_12T_thrombin_HD22_29mer; 40: thrombin_AYA1809004_40mer_8T_DBCO; 29 Click 1: 29+nPPVp* AU without heating; 29 Click 2: 29+nPPVp* AU with heating; 29 Click 3: 29+nPPVp* SU without heating; 29 Click 4: 29+nPPVp* SU with heating; 40 Click 1: 40+cPPVp* AU without heating; 40 Click 2: 40+cPPVp* AU with heating; 40 Click 3: 40+cPPVp* SU without heating; 40 Click 4: 40+cPPVp* SU with heating.
Streptavidin-biotin interactions based crosslinking
Given the challenges associated with the expression of proteins incorporating unnatural amino acids, we are exploring natural bioaffinity systems that can be constructed using simply fused proteins. We have selected streptavidin-biotin interactions for this purpose. Streptavidin (SA) is a tetrameric protein derived from the bacterium Streptomyces Avidini, which exhibits extraordinary affinity for biotin. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. This approach simplifies the process while maintaining effective binding and functionality.
Figure 24: Concept diagram of crosslinking between an aptamer and split protease via streptavidin-biotin interactions. The split protease is fused with streptavidin (SA), while the aptamer is biotinylated, allowing for a strong and specific binding that enhances protease functionality.
Maltose-binding protein (MBP) tag is a large (43 kDa) periplasmic and highly soluble protein of E. coli that acts as a solubility enhancer tag. It also increases protein expression levels. We added this tag to our protein to facilitate later expression and purification. Besides, considering of the large molecular weight of MBP, we were concerned that the MBP tag may interfere with the binding of SA and biotin. So we added a TEV-cutsite between the MBP and SA.
Figure 25: Overview of plasmid construction for streptavidin-biotin interactions based crosslinking.
After performing PCR on the target genes and the pET28a plasmid backbone, Gibson assembly will be conducted to generate the desired plasmids. Then we sent these plasmids to sequence and ultimately confirmed that they were correct.
Next, we transferred the correct plasmid into the expression strain of E.coli BL21(DE3). When the culture reached an OD600 of approximately 0.55 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C.Then we did Protein Solubility Verification. We found that our protein was predominantly located in the inclusion bodies, but a portion was also present in the supernatant. The we used nickel beads to purify protein from supernatant. However, there were little target protein in eluate.
So, we can only obtain our protein from the inclusion bodies. Since the majority of the protein in the inclusion bodies is our target protein, We directly denatured the washed inclusion body protein using 8M urea, followed by gradient dialysis for refolding. Then we cleaved the refolded protein with TEV overnight.
Figure 26: SDS analysis of the protein purified from supernatant and washed inclusion bodies for SA_cPPVp, SA_nPPVp, MBP_SA_cPPVp and MBP_SA_nPPVp. Lane1-4, protein purified from supernatant. From lane 1 to 4, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively. Lane5-8, washed inclusion bodies. From lane 5 to 8, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively.
We added different amounts of cleaved MBP_SA_nPPVp to 5’biotin-29mer and MBP_SA_cPPVp to 5’biotin-40mer. After incubating at 37°C for half an hour, we performed EMSA to verify the link between protein and nucleic. The EMSA results showed that as the amount of protein increased, the nucleic acid bands darkened. However, no shifted bands were observed.
Figure 27: EMSA analysis of the protein-nucleic linkage. Lane1-4, 5’biotin-29mer and cleaved MBP_SA_cPPVp. Only add aptamer in lane 1. From lane 2 to 5, protein amounts for lane are 0.3ug, 0.6ug, 1.2ug, 2.4ug, respectively. Lane6-10, 5’biotin-40mer and cleaved MBP_SA_nPPVp. Only add aptamer in lane 6. From lane 7 to 10, protein amounts for lane are 0.9ug, 1.8ug, 3.6ug, 7.2ug, respectively. The amounts for aptamer is 20pmol each lane.
Our experimental results preliminarily demonstrated the binding capability between SA-split protease and biotin-aptamer. However, it is strange that no band shift was observed. We suspect this is because the refolded inclusion body protein remains partially aggregated, causing it to get stuck in the wells during gel electrophoresis and not run down. Due to time constraints, we did not conduct further experiments to verify the linkage.
Module IV: Gold Colloid-based Output
In this module, β_hCG_GST works as our output signal. Firstly, the substrate of PPVp is designed as a fusion protein(GST–PPVp cleavage site–hCG), and the substrate is immobilized on a GST affinity chromatography column. Then, when our sample flows through the column, if the split-PPVp is active in the sample, cleavage happens. hCG flows out into the hCG colloidal test paper. The test paper will show a positive result. If there isn’t any active PPVp in the sample, then the hCG colloidal test paper will show a negative result.
Figure 28: Concept diagram of realizing output with hCG colloidal gold test paper. Fusion protein (GST—PPVp site—hCG) is immobilized on a GST affinity column. Active PPVp in the sample cleaves the substrate, releasing hCG, which is detected by the hCG colloidal test paper, showing a positive result. No cleavage results in a negative test.
First, we constructed pET42a_β_hCG_GST, then we sent the plasmid to sequence and ultimately confirmed that it was correct(Fig 1).
Figure 29: Construction of pET42a_β_hCG_GST
The plasmid was transformed into BL21(DE3) and induced with IPTG. SDS-PAGE was performed to verify the expression of target protein, and we can see a band on the correct position.
hCG colloidal test paper was acquired to test the biological activity of β-hCG. Unfortunatly, the result was negative. After investigation, we found out that though the specificity of hCG was determined by its β subunit, most kinds of the colloidal test paper require both α and β subunits to fully perform their function. Our future plan is to express the fusion protein of α_β_hCG_GST.