Part:BBa_K4195087

myc tag-pirA-his

Biology

PirA

PirA (a 111-residue protein) and PirB (a 438-residue protein) consist of the binary photorhabdus insect-related (Pir) toxins PirAB^vp. These toxins secreted from Vibrio parahaemolyticus cause an emerging disease called acute hepatopancreatic necrosis disease (AHPND) directly in shrimp aquaculture. PirA was reported to facilitate target-specific recognition by binding to certain ligands on the cell membrane/receptor (1).

Usage and design

In order to test the binding ability of purified receptors that we chose to purified toxins, a Myc-tag (EQKLISEEDL) was fused to the N-terminal of PirA-his. This second label (Myc-tag) was added to distinguish the proteins that were used to incubate with the nitrocellulose membrane from those spotted on the membrane when performing dot blot, since the proteins we purified were all fused with His-tag. We constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into into E. coli BL21(DE3). Then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. Identification

After transforming the plasmid into E. coli1(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (588 bp) can be observed at the position between 750bp and 500 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195087_pET-28a(+).

2. Protein purification of the toxins

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of Myc-PirA-his (Fig. 2), the target protein (15.8 kDa) can be observed at the position between 25 kDa and 14 kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (15.8 kDa) can be observed at the position between 25 kDa and 14 kDa.

Reference

1. S.-J. Lin, K.-C. Hsu, H.-C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirA^vp and PirB^vp Toxins. Marine Drugs. 15, 373 (2017).

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 163
  
• 1000
  COMPATIBLE WITH RFC[1000]