Part:BBa_K5531001

PPG-P15VP4

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 461
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

BBa_K5531001 (PPG-P15VP4)

Profile

Name: PPG-P15VP4
Base Pairs: 1609 bp
Origin: Eukaryotes; synthesized
Properties: The basic structure of collagen sequences involves repeating tripeptide units. The most common pattern is Gly-X-Y, where Gly stands for glycine, and X and Y can be any amino acid, though Y is typically hydroxyproline (Hyp) or proline (Pro).

Usage and Biology

The basic structure of collagen sequences involves repeating tripeptide units. The most common pattern is Gly-X-Y, where Gly stands for glycine, and X and Y can be any amino acid, though Y is typically hydroxyproline (Hyp) or proline (Pro). The collagen sequence can be represented as Gly-Pro-Hyp or Gly-X- (where Y is often Hyp, and X and Y are commonly Pro and Hyp). This repeating tripeptide unit is characteristic of collagen's structure and is crucial for its functionality and stability [1].

Fig. 1. PPG-P15VP4 Gene map

Experimental Approach

We isolated pET-Dual-N-His-TEV vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis result displayed consistency toward the expected outcome (HisPPG-P15VP4 is 2000 bp and P4H is 630 bp), indicating the success in pET-Dual-HisPPG-P15VP4-P4H construction. We selected colonies and sent them directly for sequencing. Figure 2 shows the success of pET-Dual-HisPPG-P15VP4-P4H construction.

Fig. 2. The results of pET-Dual-HisPPG-P15VP4-P4H

Later, the plasmid mounted with the gene of PPG-P15VP4 was transferred to E. coli DH5α to replicate. The extracted plasmid was transferred into E. coli BL21, which can help express His-PPG-P15VP4. After the colony PCR of E. coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression. The protein expressed via E. coli BL21 was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis. The target protein PPG-P15VP4 has a size of 59 kDa (Figure 3).

Fig. 3. His-PPG-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

References

[1] Jalan AA, Demeler B, Hartgerink JD. Hydroxyproline-free single-composition ABC collagen heterotrimer. J Am Chem Soc. 2013 Apr 24;135(16):6014-7. doi: 10.1021/ja402187t.