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Latest revision as of 03:53, 30 September 2024

_ PKG50-P4VP4



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1141
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


BBa_K5531003 (PKG50-P4VP4)

BBa_K5531003 (PKG50-P4VP4)

Profile

Name: PKG50-P4VP4
Base Pairs: 1899 bp
Origin: Self-assembled heterotrimeric collagen; synthesized
Properties: Proline-free heterotrimeric collagen-like motifs ((PKG)n, (DKG)n, (EPG)n) were expressed by fusion of three different serological Vp4 proteins (P4, P8, P15) with one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed to form oligomers in vitro [1,2].

Usage and Biology

Proline-free heterotrimeric collagen-like motifs ((PKG)n, (DKG)n, (EPG)n) were expressed by fusion of three different serological Vp4 proteins (P4, P8, P15) with one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed to form oligomers in vitro [1,2].

Figure 1: PKG50-P4VP4 Gene map
Fig. 1. PKG50-P4VP4 Gene map

Experimental Approach

We isolated pET-24a 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 (PKG50-P4VP4 is 2000 bp); we selected colonies and sent them directly for sequencing. Figure 2 shows the success of pET24a-PKG50-P4VP4 construction.

Figure 2: The results of pET24a-PKG50-P4VP4
Fig. 2. The results of pET24a-PKG50-P4VP4

Later, the plasmid pET-24a-PKG50-P4VP4 was transferred to E. coli DH5α to replicate. The extracted plasmid was transferred into E. coli BL21, which can help express His-PKG50-P4VP4. 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 PKG50-P4VP4 has a size of 68.1 kDa.

Figure 3: His-PKG50-P4VP4 using E. coli BL21. SDS-PAGE gels showing the purification results.
Fig. 3. His-PKG50-P4VP4 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] Gauba V, Hartgerink JD. Self-assembled heterotrimeric collagen triple helices directed through electrostatic interactions. J Am Chem Soc. 2007 Mar 7;129(9):2683-90.
[2] Liu Zezhong; Zhou Jie; Zhu Yun; Lu Lu; Jiang Shibo; School of Basic Medical Sciences, Fudan University; Department of Pharmacology, School of Pharmacy, Fudan University; Institute of Biophysics, Chinese Academy of Sciences.