Part:BBa_K4195090

ttpB-his-myc tag

ttpB-his-myc tag can interact with protein Vp0980 and Myc mouse antibody.

Biology

TTPB

TTPB is tail tubular protein B of podophage 7. It has been found that TTPB serves as ligands that recognizes the conserved Vibrio receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of Vibrio parahaemolyticus and then mediates phage adsorption and subsequent bacterial lysis(1).The his-tag (6×His) is added to C-terminal for protein purification.

Usage

In order to get the binding ability of purified Vp0980 to purified ttpB receptor, a Myc-tag was fused to the C-terminal of ttpB-his. We constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli DH5α & E. coli BL21(DE3). The positive transformants were selected by kanamycin, colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis After transforming the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. The expected result was obtained (2546 bp).

Fig. 1 The result of colony PCR.

2.SDS-PAGE The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by IPTG, 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 ttpB-his-myc tag (Fig. 2), the target protein (87.6 kDa) could be observed at the position around 80 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 (87.6 kDa) can be observed at the position around 80 kDa.

Reference

1.M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerg Microbes Infect. 9, 855-867 (2020). Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2112
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 765
  Illegal AgeI site found at 1090
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1665