Cultivation, Purification and SDS-PAGE

Induction Condition

Basic Silinker (BS) is a novel recombinant protein that efficiently attaches to the surface of silicon dioxide. The presence of mSA often leads to the formation of inclusion bodies, increasing the difficulty in purification. To achieve efficient expression of Basic Silinker and minimize the formation of inclusion bodies, we screened the induction conditions using IPTG. We set up a gradient of five IPTG concentrations: 0mM, 0.1mM, 0.25mM, 0.5mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 1mM. Furthermore, we tested two temperature gradients for induction: 37°C and 16°C. At 37°C, the protein mainly formed inclusion bodies rather than soluble proteins. Therefore, we determined that 16°C was the effective induction temperature.

To facilitate proper folding of mSA and reduce inclusion body formation, we modified the protein buffer by incorporating biotin. The binding of biotin to mSA helps with the correct folding of the Basic Silinker protein, minimizing the formation of misfolded inclusion bodies. As a result, we obtained soluble proteins that could be extracted from the supernatant. The formulation of the buffer and experimental procedures can be found in the reference (protocol).

Figure 1| SDS-PAGE analysis of Basic Silinker protein with IPTG concentration gradient induction. An IPTG concentration gradient of 1mM, 0mM, 0.1mM, 0.25mM, and 0.5mM was used for 16 hours induction at 16°C. A 10-190kDa Blue Plus V Protein Marker was used for protein size comparison. The lane bands in the image, from left to right, represent the marker, 1mM IPTG induction, 0mM IPTG induction, 0.1mM IPTG induction, 0.25mM IPTG induction, 0.5mM IPTG induction, and bacterial pellet. The gel was run at 80V for 10 minutes and then at 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue dye.

Purification of Basic Silinker

After successfully determining the expression conditions for the BS protein, it is necessary to scale up the culture and perform purification. We induced the expression using 1mM IPTG and harvested a large amount of the target protein after 16 hours of induction at 16°C. In the process of plasmid construction, we incorporated a His tag into the target protein, which facilitated purification using nickel affinity chromatography based on the specific binding of the His-tagged protein. The results, as shown in the figure 2, demonstrate the successful elution of a large amount of the target protein using 400mM imidazole, indicated by distinct bands. Although inclusion bodies were still formed, they can still meet the requirements for subsequent experiments.

Figure 2| SDS-PAGE analysis of Basic Silinker protein purification. Large-scale expression of Basic Silinker protein was conducted at 16°C with 1mM IPTG induction, followed by purification using nickel affinity chromatography. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 9, they represent the flow-through, 50mM imidazole elution 1, 50mM imidazole elution 2, 50mM imidazole elution 3, 400mM imidazole elution 1, 400mM imidazole elution 2, 400mM imidazole elution 3, 1M imidazole elution 1, and 1M imidazole elution 2, respectively. The gel was run at 80V for 10 minutes and then at 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue dye.

Purification process optimization

Due to the addition of biotin in the protein purification process to assist with protein folding and considering that endogenous biotin may occupy the mSA site, the presence of biotin in the solution can affect the availability of open mSA binding sites. Therefore, it is necessary to remove biotin from the solution. In order to improve the purification strategy, we have also developed corresponding hardware to aid in the purification process. Firstly, we employ thrombin enzyme to cleave the trxA-His tag, exposing the mSA site. The protein mixture is then incubated with silica gel beads for 1 hour, followed by washing the beads 5 times with 100 mM Tris-HCl buffer (pH 8.0) at 75°C. Subsequently, the protein is eluted using a mixed buffer containing 2M arginine, 700mM NaCl, and 0.3% Tween 20 (pH 9), resulting in the obtainment of Basic Silinker protein with available open binding sites. For more detailed information regarding the hardware, please refer to the

Figure 3| SDS-PAGE analysis of Basic Silinker protein purification using SiO₂ column. Lane 1-3 represent Basic Silinker elution flowthrough, 2M Lys wash, 1M Lys wash, 0.5M Lys wash, The gel was run at 80v for 10 minutes followed by 150v for 20 minutes, stained with Coomassie Brilliant Blue, and subjected to protein gel analysis.

Figure 4| SDS-PAGE analysis of Basic Silinker protein purification using SiO₂ column. NaCl was further added to the solution of 2M lysine. Lane 1-5 represent Basic Silinker elution flowthrough, 50mMNaCl wash, 100mMNaCl wash, 300mMNaCl wash, 500mMNaCl ,700mMNaCl wash. The gel was run at 80v for 10 minutes followed by 150v for 20 minutes, stained with Coomassie Brilliant Blue, and subjected to protein gel analysis.

Figure 5| SDS-PAGE analysis of Basic Silinker protein purification using SiO₂ column. Further pH adjustment was performed in a solution of 700mMNaCl, 2M lysine. Lane 1-4 represent Basic Silinker elution flowthrough, pH=7.5 wash, pH=8.0 wash, pH=8.5 wash, pH=9.0 wash. Lane 5 Elution was carried out in a solution of 700mMNaCl, 2M lysine, pH=9.0 with the addition of an additional 0.3%Tween. The gel was run at 80v for 10 minutes followed by 150v for 20 minutes, stained with Coomassie Brilliant Blue, and subjected to protein gel analysis.

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Structure and Biological Activity Analysis

Ultraviolet Spectroscopy

The spectra generated by peptide bonds in different protein or peptide secondary structures exhibit distinct band positions and absorption intensities. Consequently, we can determine the secondary structure of a protein based on the information provided by its far-ultraviolet (UV) spectroscopy. To perform the analysis, Basic Silinker and mSA were dissolved in 1×PBS containing various concentrations (0-10M) of guanidine hydrochloride (GdnHCl) to achieve a final concentration of 0.01mg/ml. The fluorescence emission spectra of the sample were recorded using a 295 nm excitation wavelength, a 1 nm emission bandwidth, and a scan speed of 100 nm/min at room temperature (approximately 25°C). The measurements were conducted in a cuvette with a 1 cm pathlength.

mSA exhibits stability at high temperatures. Hence, conducting thermal unfolding studies to compare the stability of mSA and Basic Silinker is challenging in practice. Instead, we employed fluorescence spectroscopy to comparatively assess the relative stabilities of mSA and Basic Silinker in the presence of a chemical denaturant, GdnHCl, which induces denaturation of both mSA and Basic Silinker. While mSA contains three tryptophan residues, Basic Silinker lacks any of these aromatic amino acids present in the mSA domain. To minimize the excitation of tyrosine residues, which usually occurs between 280 and 290 nm, our fluorescence experiments were performed using a 295 nm excitation wavelength. The overall fluorescence of mSA and Basic Silinker can be attributed to the tryptophan residues in mSA. Tryptophan fluorescence is sensitive to the environment, and the wavelength of maximum fluorescence emission is associated with the folding state of the protein, with hydrophobic environments exhibiting fluorescence at longer wavelengths. Assessing the properties of mSA and Basic Silinker proteins involved monitoring the maximum tryptophan fluorescence emission wavelength shift towards longer wavelengths with increasing GdnHCl concentration.

The GdnHCl denaturation curves of mSA and Basic Silinker are depicted in Figure 1. The changes in relative fluorescence intensity at 330 nm and 360 nm (330/360) were monitored to observe variations in the maximum fluorescence emission upon excitation at 295 nm. The tryptophan fluorescence remained unchanged until approximately 4M GdnHCl for both mSA and BS. Similarly, both proteins exhibited maximum unfolding at around 6M GdnHCl. These results indicate that mSA and Basic Silinker possess similar chemical stabilities, signifying that the connecting region of Basic Silinker does not significantly affect the structural stability of mSA.

Figure 1| mSA （blue） and Basic Silinker （red） were denatured in guanidine hydrochloride and diluted in 1×PBS containing 0-10M guanidine hydrochloride to a final concentration of 0.01 mg/ml.

Circular Dichroism Spectrum

Proteins are multi-level structures formed by the linkage of amino acids through peptide bonds. The peptide bonds, aromatic amino acid residues, and disulfide bridges in the structure are all optically active functional groups. Moreover, the optical activity of proteins is influenced by their secondary and tertiary structures. This phenomenon is known as protein circular dichroism (CD), which follows certain patterns in CD spectra. PBS strongly absorbs at wavelengths below approximately 200 nm, which prevents the collection of CD data at these wavelengths. Therefore, all CD data were collected in water.

Wavelength scans were conducted between 180 and 350 nm using a rectangular, 1 mm pathlength quartz cuvette. For each sample, three accumulations were recorded with a 2 nm bandwidth, a scan speed of 100 nm/min, and a digital integration time (DIT) of 2 s. The data are presented in terms of mean residue ellipticity (θM), expressed in deg·cm2·dmol−1·residue−1.

Figure 2| The compositional analysis of the secondary structure of BS

Figure 3| Far-UV spectra of Basic Silinker in water at a concentration

Figure 4| The compositional analysis of the secondary structure of mSA

Figure 5| The compositional analysis of the secondary structure of mSA

We are focusing on the β-sheet segments of the protein because the functional structure of the protein is a barrel-shaped β-fold structure in the mSA region, as shown in Figure 9. In this figure, all parts of the mSA segment are of the strand1 secondary structure. However, the bs segment has 0.17% of peptide segments. We speculate that this is because the SBP sequence is a peptide segment without secondary structure. However, the sequence lacks alpha helix, which we speculate is due to incomplete removal of the Trx tag, resulting in a very low proportion of alpha helix. It can be observed that in the secondary structures of both proteins, β-sheets dominate the main segments, indicating that the mSA region still retains its original biological activity.

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Functional Testing

Basic Silinker Helps to Modify Protein to the Silica Surface

Verification of SBP Binding to Silica Surface

To confirm the binding ability of Basic Silinker protein to the surface of silicon dioxide, we conducted a co-incubation experiment of Basic Silinker with silicon dioxide and analyzed the results using SDS-PAGE. The results are shown in the figure 1. We observed that both Basic Silinker and miscellaneous proteins were abundant in the supernatant. However, with each successive wash, the protein bands corresponding to Basic Silinker became progressively lighter, indicating that most of the unbound protein was washed away. To further confirm the binding, we subjected the protein to denaturation using heat and denaturing agents, causing the Basic Silinker to dissociate from the silicon dioxide surface. As a result, we observed protein bands corresponding to Basic Silinker using both denaturation methods, but significantly fewer bands were visible after heat denaturation, suggesting that some Basic Silinker lost its activity through heating. Based on these findings, we can conclude that Basic Silinker is capable of binding to the surface of silicon dioxide.

Figure 1| SDS-PAGE analysis of the in vitro co-incubation of purified Basic Silinker protein with SiO2. A protein ladder, Blue Plus V Protein Marker with a range of 10-190 kDa, was used for comparison. Lanes 1-6 in the figure represent the supernatant, SiO₂ washed once, SiO₂ washed twice, SiO₂ washed three times, incubated at 99°C with SiO₂ for 20 minutes with loading denaturing buffer, and incubated at 99°C with SiO₂ for 20 minutes without loading denaturing buffer, respectively. Electrophoresis was performed at 80 V for 10 minutes, followed by 150 V for 20 minutes. The gel was stained with Coomassie Brilliant Blue for subsequent analysis.

Verification of mSA Binding to Proteins

We also want to verify the successful connection between the biotinylated target protein and the mSA of the Basic Silinker, thereby completing the protein modification on the surface of silica dioxide. To do this, we chose bovine serum albumin (BSA) for simulation. Firstly, we biotinylated the BSA protein (biotin-BSA), and then cleaved the trxA tag of the Basic Silinker protein to expose the mSA site. Next, we connected the cleaved Basic Silinker to the silica dioxide surface and co-incubated it with biotin-BSA for 3 hours. After that, the silica dioxide was washed three times with elution buffer to remove any unbound proteins. Finally, the entire protein system was denatured to verify the connection status.

Figure 2| Verification of the connection between Basic Silinker protein and biotinylated target protein. Biotinylated BSA protein was incubated with purified TrxA protein tag-cleaved and endogenous biotin-removed Basic Silinker-SiO₂ system for 3 hours in vitro. A protein ladder ranging from 10-190 kDa using the Blue Plus V Protein Marker was used for comparison. In the figure, lanes 1-12 correspond to BSA-SiO2 running buffer, BSA-SiO₂ third elution buffer, BSA-SiO₂ first elution buffer, BSA-SiO₂ second elution buffer, BSA-SiO₂ co-incubated at 99℃ with loading elution buffer, BSA-BS-SiO₂ stock solution, BSA-BS-SiO₂ co-incubated at 99℃ with loading denaturing elution buffer, BSA-BS-SiO₂ second elution buffer, BSA-BS-SiO₂ third elution buffer, BSA-BS-SiO₂ first elution buffer, and Blue Plus V Protein Marker standard solution. The gel was subjected to electrophoresis at 80V for 10 minutes and 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue and protein gel analysis.

The silica dioxide system connected with the Basic Silinker protein (excluding the trxA tag) was co-incubated with the biotinylated BSA protein, and the protein components were identified by SDS-PAGE and Coomassie brilliant blue staining. The results revealed the following: In the silica dioxide system without the Basic Silinker connection, BSA was released from the silica dioxide system with each cycle of washing. Additionally, after the addition of denaturing agent, there was no corresponding band for BSA, indicating the absence of BSA protein in the system and the unsuccessful protein modification on the silica dioxide surface. On the other hand, in the silica dioxide system connected with Basic Silinker, BSA was successfully attached to the silica dioxide surface, with only a small amount of BSA protein detected in the elution buffer. After denaturation, a significant amount of BSA protein was washed off, confirming the successful modification of BSA onto the silica dioxide surface. This validates the success of our design.

Visualization of Silica Surface Protein Modification

In order to confirm whether Basic Silinker can successfully function in the modification of functional proteins onto the surface of silica dioxide, we used a mutant variant of green fluorescent protein (eGFP) as a simulation for functional protein modification, providing a visual representation of the connection results. We deposited both the Basic Silinker-modified eGFP (BS-eGFP) and the unmodified eGFP onto a microscope slide.

Next, we used a standard pipette tip as a pen to write the two kinds of proteins on the surface of the slide. As shown in the figure 3, a single washing step removed the eGFP protein from the surface, but it left behind a layer of BS-GFP. Additionally, when washed with a 2M l-Lys solution (700mM NaCl, 0.3% Tween, pH=9.0), the protein was eluted. The high salt and high pH condition of l-Lys serve as an effective elution buffer for the protein.

Figure 3| Deposit 10 μL of BS-eGFP as well as eGFP onto a clean microscope slide. After a 10-minute incubation, observe the sample. For the washing step, since the slide has already dried, and the protein is in a crystalline state, first soak the slide in water for 1 minute, then wash for 30 seconds, repeating the washing step 2 times.

Thermal Stability of SBP Sequences

In order to remove endogenous biotin and expose the binding site of mSA, our designed Basic Silinker is theoretically a highly heat-stable protein. To validate our design, we performed elution experiments under different temperature gradients (room temperature, 40°C, 50°C, 60°C, 75°C, 80°C, 99°C) to verify the thermal stability of the Basic Silinker.

It can be observed that no apparent target bands were detected under all temperature gradients.

Figure 4| In vitro co-incubation and thermal stability verification of Basic Silinker protein with SiO₂. The purified Basic Silinker protein was co-incubated with SiO₂ for 3 hours in vitro, followed by elution experiments under different temperature gradients. The protein ladder was compared using the Blue Plus V Protein Marker ranging from 10-190kDa. In the figure, lanes 1-8 represent the following: co-incubation with SiO₂ at room temperature for 10 minutes without loading elution medium, co-incubation with SiO₂ at 40°C for 10 minutes without loading denaturing elution medium, co-incubation with SiO₂ at 50°C for 10 minutes without loading denaturing elution medium, co-incubation with SiO₂ at 60°C for 10 minutes without loading denaturing elution medium, co-incubation with SiO₂ at 75°C for 10 minutes without loading denaturing elution medium, co-incubation with SiO₂ at 80°C for 10 minutes without loading denaturing elution medium, co-incubation with SiO₂ at 99°C for 10 minutes without loading denaturing elution medium, and Basic Silinker protein solution with TrxA protein tag removed. The gel was run at 80V for 10 minutes followed by 150V for 20 minutes, stained with Coomassie Brilliant Blue and analyzed.

To exclude the possibility of unsuccessful incubation leading to the lack of successful binding between silica dioxide and Basic Silinker, we added denaturing agents to the residual samples before performing the elution. The results are shown in the figure 5. It can be observed that protein bands appeared at all temperature gradients without significant differences. Therefore, we can conclude that Basic Silinker is a heat-resistant and thermally stable protein, consistent with our design.

Figure 5| In vitro co-incubation and thermal stability denaturation verification of Basic Silinker protein with SiO₂. The purified Basic Silinker protein was co-incubated with SiO₂ for 3 hours in vitro, and loading denaturing agents were added for elution experiments under different temperature gradients. The protein ladder was compared using the Blue Plus V Protein Marker ranging from 10-190kDa. In the figure, lanes 1-11 represent the following: Throm-cut Basic Silinker protein solution, co-incubation with SiO₂ at 99°C with loading elution medium, co-incubation with SiO₂ at 80°C with loading elution medium, co-incubation with SiO2 at 75°C with loading elution medium, co-incubation with SiO2 at 60°C with loading elution medium, co-incubation with SiO2 at 50°C with loading elution medium, co-incubation with SiO2 at 40°C with loading elution medium, co-incubation with SiO₂ at room temperature with loading elution medium, SiO2 wash for 3 times, SiO₂ wash for 2 times, and SiO₂ wash for 1 time. The gel was run at 80V for 10 minutes followed by 150V for 20 minutes, stained with Coomassie Brilliant Blue, and analyzed.

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Cut Silinker (CS) is a new recombinant protein, in addition to connecting to the surface of silica, the core breakthrough is the ability to cope with a variety of microenvironments specific cut, release off and then play a role in the release of the drugs. CS is divided into three parts, which can be self-sheared through the intein, and finally connected together to form a complete recombinant protein.

The three components include: mSA-intein peptide (CS1) for coupling to the target protein, SBP-intein peptide (CS3) for linking to silica, and changeable recognized cleavage site (CS2). We chose the PLGVR motif, which can be recognized by Matrix metalloproteinases (MMPs) in tumor microenvironments and added intein to both ends to achieve interchangeable insertion connections. that enable interchangeable insertion junctions.

The sequences were added with HIS tags, allowing purification to be accomplished using nickel columns. A TrxA Solubilizing tag was added upstream of the sequences to aid protein folding and reduce inclusion body formation in the bacterium. After CS1 protein expression, cleavage of thrombin allows for exposure of the mSA site to bind to functional proteins that have been biotinylated proteins that have been biotinylated.

We determined the conditions for the production of His-tagged Cut Silinker by performing a small trial expression of the petDUT1 plasmid after transferring it into our engineered bacterium BL21(DE3). The purified Cut Silinker could be detected by SDS-PAGE, and the molecular weights of CS1, CS2, and CS3 were 41 kDa, 18 kDa, and 8 kDa, respectively.

Earlier our attempts were based on 37°C while IPTG remained at three gradients of 0, 0.1, and 0.25.The molecular size of CS2 is about 19 kD, but in gel results (figure 3),there is a significantly high expression of a protein around 35 kD that observation clearly contradicts the expected size of CS2.

Figure 1| Cut Silinker 2 protein IPTG concentration gradient induced expression SDS-PAGE plot. Expression was induced at 37°C for 6 h. Protein scales were compared with rainbow Marker at 20-245 kDa. SDS-PAGE gels were put into runningbuffer successively electrophoresed at 80v for 30 min and 120v for 120 min, and stained with Coomassie Brilliant Blue Stain, and then analyzed as protein gels afterward.

For CS3, under the same induction conditions, we can still observe the existence of a protein expression of about 25kD(figure 2), but our protein CS3, about 8kD does not have an obvious band, this may suggest that the temperature of our induction is not appropriate.

Figure 2| SDS-PAGE of expression induced by the concentration gradient of IPTG of Cut Silinker 3 protein. The expression was induced at 37℃ for 6 hours, and the protein scale was compared with 20-245kDa Blue Rainbow Marker. SDS-PAGE glue was put into running buffer for 80v electrophoresis for 30min, 120v electrophoresis for 120min, and dyed with Coomasil bright blue solution, and then protein glue analysis was performed. We can still observe the existence of a protein expression of about 25kD, but our protein CS3, about 8kD does not have an obvious band.

Therefore, we changed the temperature to 16℃ and induced expression for 16h. In our gel results（figure 3），CS2 protein is obviously expressed, but most of the protein is insoluble and formed inclusion bodies in the precipitation, but it is rarely expressed in the supernatant.

Figure 3| SDS-PAGE of expression induced by the concentration gradient of IPTG of Cut Silinker 2 protein. The expression was induced at 16℃ for 16 hours, and the protein scale was compared with rainbow Marker of 25-245 kda. SDS-PAGE glue was put into runningbuffer for 80v electrophoresis for 30min, 120v electrophoresis for 120min, and dyed with Coomasil bright blue solution, and then protein glue analysis was performed.

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After successfully obtaining Cut Silinker proteins, the target proteins need to be cultured and purified in large quantities. We followed a 0.25 mM IPTG concentration for inducing large-scale expression and obtained a significant amount of target protein after 16 hours of induction at 16°C. During the plasmid construction process, we incorporated a His tag onto the target protein, which allowed us to purify it using nickel column affinity chromatography based on the specificity of His tag protein.

Figure 4| Cut Silinker 1 protein purification SDS-PAGE plot. Cut Silinker protein mass expression was performed at 16°C and 0.25 mM IPTG concentration, followed by purification by nickel column affinity chromatography. The protein scale was Solarbio PR1930 Rainbow 245plus Broad Spectrum Protein Marker (5-245KD) . Lanes 1-8 in the figure are rainbow marker, rainbow marker, CS1 supernatant, CS1 flow-through solution, 10 mM imidazole eluent, 40 mM imidazole eluent, 100 mM imidazole eluent, and 200 mM imidazole eluent, respectively. It was successively electrophoresed at 80v for 30min and 150v for 60min, and stained with Coomassie Brilliant Blue staining solution. There were target bands in the supernatant and through-flow solution, but not in the eluate.

In order to increase the amount of protein extraction, we performed CS1 1L system expression, and used buffer containing biotin and PBS respectively during sonication, and the gel run results showed that the protein content in the supernatant of both systems was similar. In view of the difficulty in extracting CS1,CS2, and CS3 separately during the induction phase, we tried to make the three CS proteins co-incubated as a complete Cut Silinker during cell fragmentation to reduce the difficulty in extraction and purification.We conducted CS1, CS2, and CS3 200mL system expression, and tried to co-incubate the three systems. The 200 mL inducible expression system of the three proteins was subjected to bacterial fragmentation and centrifugation and the supernatants were all added to the co-incubation system for co-incubation, and no further separate SDS-PAGE was performed. The results of gel running showed that there were no obvious bands before and after the filter.

Figure 5| CS1 and Co-incubation System 4-20% Separation Gel SDS-PAGE. As shown in the figure, the lanes are, from left to right, protein marker, blank control, CS1 protein extraction supernatant under PBS Buffer, CS1 protein extraction precipitate under PBS Buffer, CS1 protein extraction precipitate under Biotin Buffer, biotin Buffer, CS1 protein extraction precipitate under PBS Buffer, biotin Buffer, PBS Buffer under PBS Buffer to extract the supernatant of CS1 protein, Biotin Buffer under PBS Buffer to extract the precipitate of CS1 protein, CS123 protein co-incubation, CS123 protein co-incubation. It can be seen that the supernatant of CS1 extracted under PBS Buffer contains less CS1, and the precipitates contain relatively more CS1 but at the same time a large amount of heterogeneous proteins. Biotin Buffer was slightly more effective than PBS Buffer, but also faced the problem of inclusion body precipitation and the difficulty of too much heteroprotein. Co-incubation prior to protein purification showed that the bands were difficult to visualize due to the excessive amount of heteroproteins.

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Structure Simulation

In view of the difficulty of purification, we predicted the structure of the three parts of the cuttable silinker protein by alphafold2 and analyzed it.

For the structure of CS1, mSA was connected to the N-terminus of the intein GP41 through the flexible linker of (GSSSS)12. It can be seen that the structure of the mSA in the front part is relatively stable, showing a barrel-like structure, and there exists a certain distance from the intein, which is not easy to have an effect on the purification; however, the His tag and the trxA of the auxiliary purification are stacked on top of each other, and the overall structure may make it not easy to be better adhered when passing through the Ni-EDTA column. For the intein part, the rigid linker fully isolated the intein and mSA, so that the intein was fully unfolded without mutual interference with other domains, which was conducive to the mutual adsorption and connection of intein.

Figure 6| Structural prediction results of Cut Silinker protein 1, blue color is relatively high fit, while red color is relatively low fit, in between is medium confidence.

Similarly, Cut Silinker protein 2 presents an enveloped structural state, where the two parts of the intein overlap with each other wrapping around the possible enzyme cleavage site, and also possibly interfering with the ligand reaction, which is taken into account during the modeling process (see the modeling section for more details), but the His tag used for purification is relatively outwardly oriented, which should theoretically be advantageous for the purification, but it still has a some difficulty and may be influenced by other factors.

Figure 7| Structure prediction results for Cut Silinker Protein 2, with blue color showing relatively high degree of fit and red color showing relatively low degree of fit, with medium confidence in between.

For Cut Silinker protein 3, the structure prediction is relatively low confidence, which is due to the fact that the SBP we chose belongs to the flexible structure itself, and its properties are subjected to a relatively large concentration of solution ions. In the actual experimental process, unfortunately we were not able to develop a solution favorable for its purification, which has a lot to do with our failure in purifying the process. However, we have taken into account the variability of this factor in the modeling process, so that the variability of its interaction in the linkage reaction and contact with SIO2 has been simulated.

Figure 8| Structure prediction results for Cut Silinker Protein 3, with blue color showing relatively high degree of fit and red color showing relatively low degree of fit, with medium confidence in between.

Enzyme Digestion

Since protein purification was not successfully completed, this part of the results simulated the process using model, based on the concentration, mass, and relevant hydrophilic properties of the three proteins, as well as the Mie constants of the enzymatic reaction, and taking into account the temperature of the human body, to simulate the release of the experimental drug, as detailed in the [MODEL] section.

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To enable the self-curling functionality within the Twisted Silinker, we incorporated a combination of calmodulin-binding peptides and CaM to minimize intramolecular binding possibilities. We also designed a rigid linker that separates different functional domains, to avoid interference while extending. Additionally, the monomeric form of streptavidin offers several advantages over the tetrameric form. Therefore, we constructed a plasmid carrying a sequence in the construction of a plasmid consisting of monomeric streptavidin, calmodulin-binding peptide, rigid linker, CaM and SBP, and used Alpha Fold 2 modeling to simulate its structure, as shown in Figure 1. To directly validate the curling functionality of the Twisted Silinker, we replaced the linker sequence with GCaMP6m and simulated its structure using Alpha Fold 2, as shown in Figure 2. Finally, thorough validation of the plasmid sequence was performed, confirming accurate sequencing results.
                 

Figure 1| Twisted Silinker modeling simulation diagram (simulated by Alpha Fold 2)

                 

Figure 2| Twisted Silinker-GFP modeling simulation diagram (simulated by Alpha Fold 2)

Continuing, we transferred the pETDuet-1 plasmid from BL21 strain into competent E. coli BL21 (DE3) cells using the heat shock transformation method. The transformation was carried out on LB solid medium supplemented with Ampicillin resistance. After 13 hours, bacterial colonies could be observed, confirming the successful transformation of the plasmid.                                                      

Figure 3, 4| Twisted Silinker-GFP streaking plate

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First, a small-scale expression was performed to screen the IPTG induction conditions. Different concentrations of IPTG (0mM, 0.1mM, 0.25mM, 0.5mM) were added to the Twisted Silinker - GFP bacterial culture for induction.

                 

Figure 5| SDS-PAGE analysis of Twisted Silinker - GFP protein expression induced by IPTG concentration gradient. The bacterial culture was induced for 16 hours at 16℃ with different IPTG concentrations (0mM, 0.1mM, 0.25mM, 0.5mM). A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. From lanes 1 to 12, they represented the suspenion (0mM, 0.1mM, 0.25mM, 0.5mM), soluble fraction after cell lysis (0mM, 0.1mM, 0.25mM, 0.5mM), and insoluble fraction after cell lysis (0mM, 0.1mM, 0.25mM, 0.5mM). The SDS-PAGE gel was stained with Coomassie brilliant blue, and subjected to protein gel analysis.

There was no obvious target protein in the supernatant. The band of our target protein only appeared in the bacterial suspension (0.1mM IPTG induced).

We induced protain expression at the concentration of 0.1mM IPTG on a large scale (16℃, 20h). Then we purified it by nickel column affinity chromatography, with the help of His-tag. Result is shown in the Figure 5, a large number of target proteins were successfully extracted from 200mM imidazole elution. There was a obvious band in the Lane 6, and the size is consistent with the predicted 76kDa.

                 

Figure 6| Twisted Silinker - GFP Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker - GFP protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. From lanes 1 to 5, they represent the flow-through, 10mM imidazole elution, 40mM imidazole elution, 100mM imidazole elution, 200mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis.

We conducted large-scale induction of expression with 0.1mM IPTG concentration (16°C, 20 hours) and performed nickel column affinity chromatography purification using His-tag. The results, as shown in Figure 6, revealed the presence of a significant amount of the targeted Twisted Silinker protein in the 500mM imidazole elution. Lane 1 exhibited clear bands, with sizes matching the predicted 53kDa.

                                   

Figure 7| Twisted Silinker Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. Lane 1 represent the 500mM imidazole elution and Lane 2 represented the 200mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis.

Verification of Protein Function

To validate that the extracted protein indeed represented our designed function, we performed Western Blotting.

As both our Twisted Silinker protein and Twisted Silinker - GFP protein carry a His-tag, if the protein is indeed Twisted Silinker or Twisted Silinker - GFP, the His-tag will specifically bind to the Anti-6×His-tag monoclonal antibody. The HRP-conjugated secondary antibody, when bound to the primary antibody, will indicate the position of the primary antibody, which corresponds to the location of the studied protein, affirming the successful extraction of Twisted Silinker protein and Twisted Silinker - GFP protein.

Unfortunately, despite attempting different buffers, transfer temperatures, and developing reagents, Western Blotting still did not yield the desired results.

Verification of Protein Folding

To validate if Twisted Silinker protein undergoes conformational changes in the presence of calcium ions, we decided to perform Native-PAGE.

As Native-PAGE does not involve the use of SDS, it allows the protein to retain its natural shape and charge during the electrophoresis process. We designed a gradient of calcium ion concentrations to examine whether the presence of calcium ions affects the conformation of Twisted Silinker protein and whether further folding of Twisted Silinker protein occurs at different calcium ion concentrations. Which was disappointing was that the gel showed a blank region without any visible bands.

Verify That changes in calcium ion concentration can cause Twisted Silinker folding, and explore the appropriate concentration.

Firstly, we conducted pre-experiment to design appropriate gradients of both calcium chloride concentration and reaction time. Results of the preliminary experiment are shown below.

Figure 8 Fluorescence in RFU of 8 preset calcium chloride concentration solutions 100μL with Twisted Silinker - GFP protein solution 100μL (Blue lines refer to original data, and the orange lines refer to the revised data)

                                   

Figure 9| Fluorescence in RFU of 8 preset calcium chloride concentration solutions 100μL with Twisted Silinker - GFP protein solution 150μL (Blue lines refer to original data, and the orange lines refer to the revised data)

                 

Figure 10| Fluorescence in RFU of 8 preset calcium chloride concentration solutions 100μL with Twisted Silinker - GFP protein solution 200μL (Blue lines refer to original data, and the orange lines refer to the revised data)

Based on the preliminary experimental results, the three different ratios of calcium ions to Twisted Silinker - GFP protein did not show significant and stable fluorescence signals.

In subsequent experiments using a larger system, we set the gradient of calcium ion concentrations to be 0.02M, 0.1M, and 0.5M, and the gradient of Twisted Silinker - GFP protein solution volumes to be 100μL, 150μL, and 200μL. The fluorescence emission of Twisted Silinker - GFP protein samples were incubated with calcium ion solution for 20s, 1min, and 15min was tested.

During the incubation, the values of the experimental group and the control group were similar, and the values of the experimental group did not show significant changes during testing. We considered variables such as reaction time, temperature, calcium ion concentration, and buffer solution in multiple controlled experiments. But unfortunately, we were unable to obtain the desired results. Therefore, it is regrettable that we cannot conclude that Twisted Silinker - GFP protein can fold in the presence of calcium ions.

                                   

Figure 11| Fluorescence in RFU of blank

                                   

Figure 12| Fluorescence in RFU of Experimental group Black lines refer to original data, and the yellow lines refer to the revised data (based on the blank groups)

Verification of correct combination of target protein, Twisted Silinker and silica particles.

First, we conducted an experiment to remove the TrxA tag from Twisted Silinker - GFP protein through thrombin cleavage, exposing the mSA site. To find the appropriate cleavage system, we chose buffer solutions of 600μL, 400μL, and 100μL, and performed cleavage at 37℃ for 0.3 hours. Unfortunately, the results, as shown in the figure 13, displayed multiple protein bands, but none of them matched the correct size.

Figure 13| Results of thrombin cleavage of Twisted Silinker - GFP. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190kDa. Lane 1 represents the 600μL Buffer solution system, Lane 2 represents the 400μL Buffer solution system; Lane 3 represents the 100μL Buffer solution system.

                 

First, we conducted an experiment to remove the TrxA tag from Twisted Silinker - GFP protein through thrombin cleavage, exposing the mSA site. To find the appropriate cleavage system, we chose buffer solutions of 600μL, 400μL, and 100μL, and performed cleavage at 37℃ for 0.3 hours. Unfortunately, the results, as shown in the figure 13, displayed multiple protein bands, but none of them matched the correct size.

Next, we attempted to verify whether Twisted Silinker could successfully connect to the surface of silica particles. We mixed silica particles with Twisted Silinker protein and incubated them to achieve a proper connection. Then, we conducted elution using PBS solution, lysine solution, and loading buffer solution.

As shown in the figure 14, the protein concentration in the elution buffer significantly decreased compared to before the incubation. There was no apparent protein band in lane 5, but a noticeable band of the target-sized protein appeared in lane 7. This indicates that Twisted Silinker successfully connected to the silica surface and the connection is tight. Neither PBS solution nor lysine solution was able to separate them.

                                   

Figure 14| Binding results of Twisted Silinker protein and silica. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190kDa. Lane 1: Twisted Silinker protein solution, Lane 2: flow-through; Lane 3-5: washed by PBS; Lane 6: washed by lysine; Lane 7: washed by loading buffer.

Summary

Through function verification experiments, we found that Twisted Silinker can connect to the surface of silicon dioxide, like Basic Silinker, which laid the foundation for its subsequent successful function. But Twisted Silinker - GFP did not folded correctly and then fluoresce in a solution with a certain concentration of calcium ions, as we expected. Considering the reaction time, temperature, calcium ion concentration, buffer and other variables, we carried out several control variable experiments, but failed to detect the ideal value.

This may be due to the lower concentration or activity of Twisted Silinker - GFP protein we used, resulting in its weak fluorescence in calcium ion solution, and its unobvious numerical growth. A more likely hypothesis is that because calmodulin is generally synthesized by eukaryotes, our chassis organism E.coli may not be able to synthesize calmodulin with the correct spatial structure (although the molecular weight of the expressed protein appears to be correct from the protein glue analysis results). Spatial structural errors may mean that the calmodulin domain of Twisted Silinker - GFP does not sense the environmental calcium ion concentration correctly. This will cause it to have difficulty folding correctly and fluorescing as expected in a solution of calcium ions. In other words, if we change the chassis organisms to eukaryotic systems such as yeast, it is likely that we can express normal Twisted Silinker.

We attempted to do Western blot analysis many times to verify whether the protein heavily expressed by E.coli was Twisted Silinker - GFP with the correct spatial structure, but unfortunately failed.

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Our Pairing Silinker shares a structural similarity with the Basical Silinker. Therefore, when expressing the PS protein, we employed conditions similar to those used for BS and aimed to further investigate the impact of inducible expression medium on protein solubility. Experimental results indicated that the culture medium conditions did not have a significant effect on the solubility of the PS protein. Furthermore, we observed that even in uninduced engineered bacteria, our PS protein was expressed and formed inclusion bodies. This led us to suspect that leaky expression from the chosen T7 expression system may be the culprit for low solubility expression. Consequently, we opted for the BL21 (DE3) pLysS strain, a constitutively expressing T7 lysozyme strain, which effectively suppresses leaky expression from the T7 RNA polymerase expression system. Ultimately, we successfully achieved high solubility expression of the PS protein in this strain.

Cultivation, Purification and SDS-PAGE

Induction Condition

In the process of expressing the BS protein, we have already explored the influence of temperature and IPTG concentration on protein solubility expression. Given the highly similar amino acid sequence and spatial structure of PS and BS, we adopted the same strain,temperature and IPTG conditions for PS protein expression ( BL21(DE3),16°C, 0.1 mM IPTG). We further investigated the impact of inducible expression medium on protein solubility.

Studies on Monomeric Streptavidin (mSA) have highlighted the importance of buffering conditions, metal ions, and glucose in the medium for enhancing mSA solubility expression yield and the growth of engineered bacteria. Additionally, we observed that some literature suggests that medium conditions can affect protein solubility expression. Hence, we explored the effects of several common medium additives on protein solubility expression.

Figure 1| SDS-PAGE analysis of Pairing Silinker protein expression induced with different media ，induced with 0.1mM IPTG for 16 hours at 16°C.”. A 10-190kDa Blue Plus V Protein Marker was used for protein size comparison. The lane bands in the image represent the marker; 1：soluble fraction after cell lysis ;2: insoluble fraction after cell lysis；Contrl：insoluble fraction from uninduced cell；medium A: 50 mM KH2PO4, 50mM Na₂HPO₄,   25mM (NH₄)₂SO₄,and 2mM MgSO4;medium B: medium A with 50mM sorbitol;medium C: medium A with 2% alcohol;medium D: medium A with 4% glycerine. The gel was run at 80V for 10 min and then at 150V for 20 min, followed by staining with Coomassie Brilliant blue dye. “ * ”Marked as the target band size

The results in Figure 1 indicate that the addition of medium additives did not significantly improve the formation of inclusion bodies or enhance soluble expression. However, the appearance of bands in the uninduced control group serves as a reminder that leaky expression from the T7 expression system is quite pronounced, leading to substantial expression and the formation of inclusion bodies even under uninduced conditions.

Therefore, to inhibit the leaky expression caused by T7 RNA polymerase, we observed the BL21 (DE3) pLysS strain, which carries the pLysS plasmid and constitutively expresses T7 lysozyme to suppress leaky expression. T7 lysozyme can bind to T7 RNA polymerase to suppress leaky expression from the T7 expression system. After transferring the plasmid into BL21 (DE3) pLysS, we attempted the expression of the PS protein once again.

Figure 2| SDS-PAGE analysis of Pairing Silinker protein expression in BL21 (DE3) pLysS, induced at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-3: Cell samples; 4-6: Soluble fraction after cell lysis; 7-9: Insoluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

The experiments shown in FIG. 2 showed that the lysis protein buffer was not supplemented with biotin, and the protein was mainly present as a precipitate, which reminds us of the important effect of biotin in the protein lysate on protein solubility.

Figure 3| SDS-PAGE analysis of Pairing Silinker protein expression in BL21 (DE3) pLysS at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-2: Cell samples; 3-4: insoluble fraction after cell lysis; 5-6: soluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

After correctly using a protein lysis buffer containing biotin, we were pleasantly surprised to observe in the results of Figure 3 that the protein appeared in a soluble form in the supernatant after lysis. Based on this outcome, we have identified the conditions for further large-scale expression of the PS protein.

Purification of PS

After successfully determining the expression conditions for the PS protein, it is necessary to scale up the culture and perform purification. We induced the expression using 0.1mM IPTG and harvested a large amount of the target protein after 16 hours of induction at 16°C. In the process of plasmid construction, we incorporated a His tag into the target protein, which facilitated purification using nickel affinity chromatography based on the specific binding of the His-tagged protein. As shown in FIG. 4, we performed affinity chromatography using a nickel column and obtained a large amount of PS protein with good purity in the presence of 500Mm imidazole

Figure 4| SDS-PAGE analysis of PS protein purification. Large-scale expression of PS was inducted at 16°C with 0.1mM IPTG induction, followed by purification using nickel affinity chromatography. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 6, they represent : 1:supernatants after cells lysis,2:flow-through, 3-5:10mM imidazole elution , 6.500mM imidazole elution .The gel was run at 80V for 10 minutes and then at 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue dye.

From 1L of medium we obtained 50mg PS protein, which was sufficient to support our further experiments

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Functional Examination of PS Proteins

We verified the binding ability of PS to silica, and as shown in Figure 5, PS can bind silica well. Based on the effect of tween-20 on the ability of silinker to bind silica given by BS experiments, we tested the effect of adding tween-20 alone.

Figure 5| Binding results of PS protein and silica. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: PS: PS protein solution, FT: flow through, W1-3: washing by PBS, 1: W3 sample eluted with PBS+2% tween-20, 2: Sample from 1 with added protein loading buffer and boiling, 3: Sample from W3 with added protein loading buffer and boiling. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

FIG. 5 Results prove that PS protein can bind silica and is difficult to elute, and tween-20 cannot elute PS protein when used alone.We observed a prominent band at approximately 90 kDa, which was not present in the initially purified PS protein (Figure 4). We speculate that this is indicative of PS dimer formation (with monomers approximately 44 kDa each), aligning with our design expectations for PS. Therefore, we aimed to further validate the dimerization of PS in the presence of calcium ions. However, our attempts to separate dimers using size exclusion chromatography and obtain dimer bands using Native-PAGE yielded puzzling results. In the size exclusion experiment, we did not observe any UV absorption peak in the chromatogram, and after staining the Native-PAGE gel, there were no bands. This is a frustrating outcome, and we suspect it may be due to the inevitable presence of silica in the instruments used for both size exclusion chromatography and Native-PAGE.

To meet our goals, it implies that we must seek a nucleic acid-protein linkage other than biotin, with relatively strong binding affinity. We have identified a method for nucleic acid-protein conjugation: TrwC is a bacterial relaxase that can covalently link to the 5' end of specific DNA sequences at its N-terminus. Therefore, we have engineered a fusion protein, mSA-TrwC.

Figure 1| Alphafold prediction model for NS

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Figure 2| SDS-PAGE analysis of NS protein purification by heat. Large-scale expression of NS was inducted at 16°C with 0.1mM IPTG induction, purified by heating for 10 minutes at 55℃. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 6, they represent : 1,2: insoluble fraction after cell lysis;3,4: heated insoluble fraction after cell lysis ;5,6: insoluble fraction after cell lysisThe gel was run at 120V for 55 minutes, followed by staining with Coomassie Brilliant Blue dye.

Because of the TrxA tag, mSA and TrwC are known to have good thermal stability. As shown in figure, bands are visible near the target after heating at 55 ° C. As shown in figure 2, NS protein was not efficiently purified by heating

Figure 3| SDS-PAGE analysis of NS protein purification. Large-scale expression of NS was inducted at 16°C with 0.1mM IPTG induction, followed by purification using nickel affinity chromatography. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 8, they represent : 1,2:flow-through; 3-4:10mM imidazole elution ; 5,6:100mM imidazole elution ; 7,8:500mM imidazole elution.The gel was run at 120V for 55 minutes, followed by staining with Coomassie Brilliant Blue dye.

The supernatant from lysed induced protein expression cultures was subjected to nickel affinity chromatography. Elution bands were observed at 100mM and 500mM imidazole, but with extremely low concentration.

The strains transformed with BL21(DE3) did not exhibit significant soluble expression of the mSA-TrwC fusion protein.

After plasmid extraction, transformation of BL21(DE3)pLysS competent cells did not yield successful clones. It is suspected that the pETDuet-1-NS plasmid is incompatible with the pLysS plasmid.

Because we could not get a large amount of high purity protein, so we can't start the functional experiment.