This year, we focused on utilizing the techniques of synthetic biology to produce an entirely new protein family. Our goal was to address the challenges associated with protein modification on silicon dioxide surfaces. Through the development of our Silinker family, we aimed to create a smart drug delivery system using mesoporous silica. This system would respond to external stimuli for enhanced drug release efficiency. For a more comprehensive understanding, please refer to our project description(description). In order to validate the effectiveness of our concept in current production and scientific research, we conducted a proof of concept work. This validation process consisted of two main parts: laboratory experiments and feedback from relevant experts and stakeholders. We have received support from experts, stakeholders, and positive results from our experiments, further affirming the potential effectiveness of our project.
To validate our hypothesis, we performed structural and bioactivity analyses of the expressed Basic Silinker protein. Protein denaturation was measured using UV excitation to detect its chemical properties, while circular dichroism spectroscopy was used for protein structure analysis.
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.
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.
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 5. 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.
In order to evaluate the affinity of Basic Silinker for silicon dioxide surfaces and confirm its ability to facilitate protein modification on such surfaces, we performed a two-step conjugation experiment. This experiment involved connecting silicon dioxide surfaces with the SBP end of Basic Silinker, as well as connecting biotinylated target proteins with the mSA end of Basic Silinker. Additionally, to gather comprehensive data for future applications, we measured the molecular dynamics of the linker protein.
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 1| 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.
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, 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 2| 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.
As introduced in our background, protein modification on silicon dioxide surfaces poses a significant challenge. To underscore the importance and feasibility of Basic Silinker in the domain of silicon dioxide surface modification , we employed Basic Silinker to modify Insulin-like growth factor onto silicon dioxide surfaces and simulated the entire drug targeting process using mesoporous silicon dioxide MSN as the carrier.（Currently underway）
