Part:BBa_K4790077

6xHis

The 6xHis tag is utilized for affinity purification of proteins. This fusion tag can bind to Ni2+-affinity resin through coordination between Ni2+ ions and histidine residues. It allows purification of the protein of interest carrying the 6xHis tag after washing and elution steps.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

iGEM24_Jiangnan-China

Group: iGEM24_Jiangnan-China
Author: Ke Tong, Yueheng Niu, Hanzhi Weng, Zhiyu Shen
Summary: New findings on the effect of PET-binding peptides on the properties of fusion proteins and different choice of purification of PET-binding peptide-containing fusion proteins.

Usage and Biology

We adopted a similar plasmid construction method to that of iGEM23_NNU-CHINA, and also added a His tag on our fusion protein to facilitate purification.

Molecular construction

The fusion protein gene was synthesised and cloned into plasmid pET-21b（with T7 promotor BBa_K4790075, lac operator BBa_K4790078, RBS BBa_K4790079, and T7 terminator BBa_K4790076) to construct recombinant plasmid pET21b-PETase-linker-PET-binding peptide-His tag.

Protein expression and purification

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for expression and cultured in a 50 mL flask to express sufficient fusion protein.

After fermentation optimization with the fusion protein 50-4-N-G4S-PETase, we determined the final concentration of IPTG to be 0.05 mmol/L. After induction, the culture was incubated at 16°C and fermented for 48 h using TB medium. After culture, the fermentation broth was centrifuged, wall-broken by high-pressure homogenization, and then centrifuged again. The fermentation supernatant and supernatant of cell fragmentation were collected as the crude enzyme.

The crude enzyme activity was measured by continuous spectrophotometry method. The reaction system was 1500 μL including 30 μL 50 mmol/L pNPB, 30 μL crude enzyme liquid and 1440 μL 100 mmol/L pH 8.0 phosphate buffer. The generation rate of p-nitrophenol during 60 s was recorded at the wavelength of 405 nm. One enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze pNPB producing 1 μmol p-nitrophenol during 60 s at 60°C.

The crude enzyme liquid of the supernatant of cell fragmentation with higher enzyme activity was subjected to Ni²⁺ column affinity chromatography.

However, during the experiment, we found that some constructed fusion proteins were difficult to be purified by Ni²⁺ columns. Instead, we chose to replace the purification method to thermal purification. We took 5 mL aforementioned crude enzyme liquid and incubated it at 50°C for 2 h, and the solution was centrifuged. Then, the supernatant was purified enzyme. For proteins with good thermal stability, the purification of target proteins could be achieved as well (Fig. 1).

Fig. 1. SDS-PAGE analysis.
M: Marker; FS: Fermentation supernatant;
PC: Precipitation of cell fragmentation; SC: Supernatant of cell fragmentation;
NP: Ni²⁺ column purified enzyme; TP: Thermally purified enzyme.

Conclusion and discussion

We are of the opinion that due to the hydrophobicity of the selected PET-binding peptide, it may have affected the protein conformation, resulting in the fusion protein being difficult to bind to Ni²⁺ ions and thus difficult to be purified.

We believe that our findings on the effect of PET-binding peptides on the properties of fusion proteins and the choice of purification of PET-binding peptide-containing fusion proteins will be useful for future iGEM teams to carry out research on binding peptides.