Usage and Biology

Figure 1. 3D simulation of Nt2RepCt protein.

Nt2RepCt

Spidroins are the primary proteins that compose spider silk, renowned for their exceptional mechanical properties, including strength, elasticity, and biodegradability. These proteins are produced in specialized glands of spiders and it can have various functions, such as web construction, prey capture, and mobility.

Spidroins are characterized by repetitive amino acid sequences that contribute to their unique structural properties. They typically consist of two main domains:

N-terminal domain (Nt): This region is involved in the initial formation of silk fibers and is crucial for the protein's solubility and stability.
C-terminal domain (Ct):This domain plays a significant role in the dimerization of spidroins and helps prevent aggregation during storage. The Ct has been shown to adopt a dimeric folding structure that is conserved across different types of spidroins, indicating a common functional role in silk formation

The repetitive sequences within spidroins often contain motifs rich in glycine and alanine, which facilitate the formation of β-sheet structures that enhance the mechanical properties of the silk fibers. [1]

NT2RepCT features a complex structure that includes both repetitive elements and unique sequences that distinguish it from other spidroins. The N-terminal domain forms amyloid-like fibrils capable of creating hydrogels, which can serve as a platform for protein immobilization. [2]

SpyTag

The SpyTag is a 13-amino-acid peptide that plays a crucial role in the SpyCatcher-SpyTag system, a powerful tool for protein engineering and conjugation. This system was developed from a modified domain of the surface protein CnaB2 from Streptococcus pyogenes, which naturally forms isopeptide bonds to aid in bacterial adhesion to host cells. The SpyTag peptide specifically reacts with the protein SpyCatcher, resulting in an irreversible covalent bond that facilitates various biotechnological applications. [3]

Part generation

We assembled this part through Golden Gate Assembly using the following parts:

BBa_J428341 (linear, digested with BsaI separately and purified from agarose gel)
BBa_J435350
BBa_J435345
BBa_K5396002
and BBa_J428069

We transformed the plasmids through electroporation into the E. coli strain DH5α and confirmed the correct assembly by Sanger sequencing.

Expression and Purification of Nt2RepCt-SpyTag

Initial purification Nt2RepCt-SpyTag

A pre-inoculum was grown overnight at 30ºC before being transferred to a larger LB culture. After reaching the desired optical density, IPTG was added to induce protein expression, which continued overnight at a lower temperature. Following harvest, the cell pellet obtained from 1 liter of culture was resuspended in 40 mL of Buffer A (pH 8.0), supplemented with 1 mM PMSF and 1 mM benzamidine to prevent protease activity.

The resuspended pellet was sonicated to lyse the cells, and the sample was centrifuged at 17,000g for 40 minutes to clarify the lysate. Protein purification was then performed using Immobilized Metal Affinity Chromatography (IMAC) on an ÄKTA system, with a Ni-column equilibrated in Buffer A.

SDS-PAGE analysis revealed the presence of bands corresponding to Spidroin’s expected size (~36 kDa) in the flow-through, indicating incomplete binding to the column.

Figure 2. Analysis of NT2RepCt-SpyTag expression and purification by SDS-PAGE.

In response to these results, we modified Buffer A, by excluding imidazole, and Buffer B (by reducing imidazole concentration) and to change the elution strategy on the ÄKTA system, opting for a direct elution with 100% Buffer B to enhance purification efficiency.

Improved Purification of NT2RepCt-SpyTag

To improve the purification of Spidroin, which predominantly appeared in the flow-through in our initial attempts, we removed imidazole from Buffer A to enhance protein binding to the Ni-NTA column. Additionally, we adjusted the imidazole concentration in Buffer B and reduced the ÄKTA flow rate to 0.75 mL/min to ensure a gentler elution, aiming to avoid fiber formation during the process.

Figure 3. Chromatogram of Nt2RepCt-SpyTag purification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The peak 2 is highlighted.

After purification, an SDS-PAGE analysis was performed, confirming that the second peak contained the majority of the Spidroin protein.

Figure 4. SDS-PAGE analysis of NT2RepCt-SpyTag expression and purification.

To further improve the purification of NT2RepCt-SpyTag, we proceeded with a second purification step, where peaks 1 and 2 were combined and re-purified using the same process as before. This additional step allowed for a more effective separation of our target protein from other proteins present in the sample, originating from the expression strain used. Consequently, we achieved a higher purity of the NT2RepCt-SpyTag protein.

Figure 5. Chromatogram of the NT2RepCt-SpyTag repurification using IMAC (Immobilized Metal Affinity Chromatography) on a Ni-column. The highlighted peak corresponds to the region with the highest concentration of the target protein.

The SDS-PAGE analysis indicated a reduction in the bands corresponding to other proteins, along with an increase in the band associated with NT2RepCt-SpyTag.

Figure 6. SDS-PAGE analysis of NT2RepCt-SpyTag repurification. The pink circle highlights the band corresponding to Spidroin, confirming its successful expression and purification.

With this confirmation, we proceeded to a third purification step using Gel Filtration (GF) to achieve a more monodisperse sample. Gel Filtration effectively separated the sample based on size, with smaller molecules eluting first and larger ones later, resulting in distinct peaks in the chromatogram.

Figure 7. Chromatogram of the gel filtration purification of NT2RepCt-SpyTag. Fractions 20-21-22-23 were collected and used for further characterization of NT2RepCt-SpyTag and subsequent hydrogel formation tests.

Based on the GF results, we selected fractions 20, 21, 22, and 23 for further analysis, as the indicated molecular size was consistent with NT2RepCt-SpyTag. An SDS-PAGE analysis was then performed on these fractions to confirm the presence and purity of the target protein.

Figure 8. SDS-PAGE analysis of Gel Filtration (GF) fractions from Nt2RepCt-SpyTag purification. The prominent bands in these lanes indicate the presence of NT2RepCt-SpyTag, with a molecular weight consistent with the expected size of the protein.

Characterization

Circular Dichroism (CD)

Nt2RepCt Before and After Heating

With the now synthesized Nt2RepCt spidroin, we evaluated its CD absorbance in two conditions: before and after a heating ramp. Since it is known hydrogel gelation when submitted to high temperatures (37 or 60ºC), it is expected for the protein to change its secondary structure when creating a new structure.

For that matter, an interesting result is shown on Figure 9. While on one hand the blue curve represents the protein before ramp, the magenta curve represents after heating, with a notable contrast when comparing each curve.

Knowing that the original secondary structure absorption happens in a range between 200 and 218 nanometers, it is possible to confirm a conformational change in the protein structure. This result indicate a possible hydrogel formation.

Figure 9. Nt2RepCt circular dichroism absorbance measured before and after heating.

If we analyze the sample appearance shown on Figure 10, we can confirm actually confirm a new structure formation. In a different phase, the protein agglomerates, creating a new layer in the solution even without the need of extremely high concentrations. In further expressions, having a higher protein volume and concentration, it will be possible to create a larger structure for water filtering.

Figure 10. Hydrogel formation in the Nt2RepCt solution.

Nt2RepCt Heating Ramp

To better assess the hydrogel formation, it is now possible to measure a heating ramp for the solution. Heated from 25 to 60ºC with an increase step of 5ºC and 6 acquisitions for each temperature, the heating ramp result can be visualized on Figure 11.

In the heating process, the protein visibly loses the secondary structure, which can be determined by the CD absorbance decrease in the 200 to 218 nanometers range. In contrast, the protein does not recover its secondary structure absorbance in the cooling process. This result shows that only a part of the absorbance is recovered, showing a non-reversible process.

This is a very important information for hydrogel creation. It is fundamental for the hydrogel structure to have great stability in the ambient temperature, without reversing to the original protein state.

Figure 11. Nt2RepCt heating ramp on the left and cooling ramp on the right.

For better visualizing this specific behavior, on Figure 12 the Nt2RepCt circular dichroism is plotted in function of a specific secondary structure absorbance wavelength, that is 210 nanometers. It is evident how the CD absorbance is not recovered with time at ambient temperature.

Figure 12. Nt2RepCt heating ramp on the left and cooling ramp on the right.

Dynamic Light Scattering (DLS) Analysis of Nt2RepCt-SpyTag Fibril Formation

Dynamic Light Scattering (DLS) was employed to analyze the behavior of Nt2RepCt-SpyTag after purification and size exclusion chromatography. DLS involves directing a laser beam at the sample and measuring fluctuations in scattered light intensity caused by the Brownian motion of the particles in solution. These fluctuations are dependent on several parameters, including particle size. By applying an autocorrelation function and specific assumptions, the average hydrodynamic diameter of the particles in the sample can be determined.

To investigate the effect of increasing temperature on the sample's structural properties, four aliquots (100 µl each) of the purified Nt2RepCt-SpyTag were prepared at room temperature (25°C). Each aliquot was then subjected to different temperature conditions: 38°C, 42°C, 46°C, and 50°C, for one hour. After this period, the samples were returned to room temperature for 30 minutes, and DLS measurements were conducted immediately afterward. For each aliquot, three complete experimental runs were performed, with each run consisting of 15 measurements lasting 10 seconds each. The timing of the measurements is critical to ensure accurate DLS results, as the fluctuations in scattering are driven by Brownian motion in solution. After obtaining the three sets of data, the results were averaged to compare particle behavior across different temperatures.

The initial hypothesis was that increasing the temperature would lead to the destabilization and unfolding of the C-terminal secondary structure of Nt2RepCt-SpyTag, resulting in the formation of β-sheet amyloid-like fibrils. It was expected that as fibrils formed, the average hydrodynamic diameter of the particles would increase.

Figure 13 presents the results (average) obtained for each temperature, while Figure 14 provides a closer look at the region corresponding to larger particle diameters. As the temperature increased, the curves shifted to the right, with the 50°C curve showing the highest intensity in the larger diameter region, indicating that fibril formation occurs with increasing temperature. Additional experiments, including Circular Dichroism and electron microscopy, were performed to corroborate and further substantiate these findings.

Figure 13. This figure shows the average hydrodynamic diameter of Nt2RepCt-SpyTag samples measured at various temperatures (38°C, 42°C, 46°C, and 50°C).

Figure 14. This figure provides a closer examination of the larger hydrodynamic diameter region from the DLS measurements of Nt2RepCt-SpyTag samples at increasing temperatures.

Scanning Electron Microscopy

Now that we were able to properly assess a structure formation after heating Nt2RepCt, it is now possible to better understand its morphology. For doing this, we used a Scanning Electron Microscope (SEM) to capture the hydrogel surface, as shown on Figure 15.

Although the drying process had damaged the hydrogel surface, its topology can still be detected. It is notable there is a presence of a protein arrangement in the surface, which is rough and irregular. This can be seen by regions that are more bulky and other that are less bulky.

From the linear structures highlighted in more bright white tons, it is possible to relate the structures to a creation of fibers. Moreover, circular structures were created, which can indicate the pore formation and compartments. This can be very useful in the context of filtering particles.

Figure 15. Hydrogel surface captured on a Scanning Electron Microscope.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 96
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 30
Illegal XhoI site found at 1148
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 96
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 96
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K5396009

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