Part:BBa_K5396009

T7-Nt2RepCt

This Nt2RepCt protein has a SpyTag, a 13-amino-acid peptide that is part of the SpyCatcher-SpyTag system, which can enable irreversible protein conjugation. This part is controlled by T7 promoter.

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

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.

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]