Part:BBa_K4623012

Pairing Silinker, dimerization in response to specific signals on the silica surface

The Pairing Silinker (PS) is a novel recombinant protein that efficiently binds to the surface of silica and undergoes dimerization in response to specific signals on the silica surface. The sequence in the FASTA format includes an added HIS tag, enabling purification of the PS protein using a nickel column. Upstream of the sequence, a TrxA** (part number)** fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin** (part number)** exposes the mSA** (part number)** site, allowing binding to a biotinylated functional protein. The SBP** (part number)** sequence can bind to the silica surface, facilitating the modification of functional proteins onto the silica surface.

After introducing the pETDuet1 plasmid into our engineered BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited high basal expression levels, leading to the formation of PS inclusion bodies in uninduced strains. Therefore, in subsequent experiments, we employed the BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. Experimental results demonstrated a significant increase in the solubility of PS.

Purified Pairing Silinker can be detected via SDS-PAGE, with a molecular weight of approximately 44 kDa, higher than the theoretical value of 34 kDa. This discrepancy may be attributed to flexible C-terminal sequences like SBP. To enhance the purification strategy, we have also developed corresponding hardware, utilizing the binding affinity between SBP and silica to purify the protein. This greatly improves the efficiency of protein production and purification, reducing costs.

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]