Part:BBa_K4623004

Basic Silinker (TrxA-His-thrombin-mSA-SBP)

Usage and Biology

Basic Silinker (BS) is a novel recombinant protein that can efficiently attach to the surface of silicon dioxide. The sequence is appended with a 6x His tag, allowing for purification using a nickel column. Upstream of the sequence, a TrxA (BBa_K3619001) fusion tag is added to aid in protein folding and reduce the formation of inclusion bodies in bacterial cells. After protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site for binding with a biotinylated functional protein. The SBP (BBa_K4623000) sequence can bind to the surface of silicon dioxide, enabling the modification of functional proteins onto the surface.

After transforming the pETDuet-1 plasmid into Escherichia coli BL21 (DE3) , we conducted small-scale expression to determine the production conditions for Basic Silinker. The purified Basic Silinker was detected by SDS-PAGE and Western Blot, with a molecular weight of 36 kDa. To improve the purification strategy, we developed corresponding hardware utilizing the binding strength between SBP and silicon dioxide, greatly enhancing the efficiency of protein production and purification. The hardware can be referenced at our wiki page.

• • characterization of BBa_K4623004:

In order to ensure that the addition of a new linker does not compromise the structural integrity, biological activity, and chemical stability of mSA, we conducted a series of tests on Basic Silinker. The test results demonstrated that Basic Silinker retains the biological activity and protein structure of the mSA domain, effectively fulfilling its intended function of biotin binding, and exhibiting excellent thermal and chemical stability. In our thermal stability test, Basic Silinker maintained its connection to silicon dioxide even at 99℃. The information obtained from circular dichroism spectroscopy and far-UV spectroscopy of the protein samples led us to conclude that the incorporation of the SBP sequence does not impact the biological activity of the mSA domain in Basic Silinker.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 445
  Illegal AgeI site found at 505
  
• 1000
  COMPATIBLE WITH RFC[1000]

Cultivation, Purification and SDS-PAGE

Induction Condition

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.

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 our protocol.

Purification of Basic Silinker

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.

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.

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 created a new, cost-effective, convenient, and reusable silica protein purification hardware utilizing the affinity between the SBP sequence and silica. It simplifies and enhances the purification process of our Silinker protein, and we have already validated its feasibility.
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 protocol.

Figure 3|SDS-PAGE analysis of Basic Silinker protein purification using SiO2 column. Lane 1-3 represent Basic Silinker elution flowthrough,2MLys wash,1MLys wash,0.5M Lys wash.

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.

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 silica packing material in our hardware can be reused multiple times, resulting in low raw material costs.

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.

Figure 6| 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.

The GdnHCl denaturation curves of mSA and Basic Silinker are depicted in Figure 6. 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.

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 7|The compositional analysis of the secondary structure of BS

Figure 8|Far-UV spectra of Basic Silinker in water at a concentration

Figure 9|The compositional analysis of the secondary structure of mSA

Figure 10|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.

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. 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 11| SDS-PAGE analysis of the in vitro co-incubation of purified Basic Silinker protein with SiO2.

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 12|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 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.

Figure 13| 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.

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, 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.

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.

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. 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 14 | 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.

Figure 15 | 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.

References

[1]Sano, T., & Cantor, C. R. (1990). Expression of a cloned streptavidin gene in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 87 (1), 142–146.

[2]Lim, K. H., Huang, H., Pralle, A., & Park, S. (2011). Engineered streptavidin monomer and dimer with improved stability and function. Biochemistry, 50(40), 8682–8691.

[3] Demonte, D., Dundas, C. M., & Park, S. (2014). Expression and purification of soluble monomeric streptavidin in Escherichia coli. Applied microbiology and biotechnology, 98 (14), 6285–6295.