Usage and Biology

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.

[1] 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.

[2] 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.

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.

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 X. 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 X. 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 X. 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 X. 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 X. 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 X. 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 Y. 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.

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

Contents