Part:BBa_K5531010

pET-Dual-HisRh3C-P15VP4-P4H

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 6594
  Illegal NotI site found at 150
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 305
  Illegal BamHI site found at 119
  Illegal XhoI site found at 354
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 324
  Illegal NgoMIV site found at 671
  Illegal NgoMIV site found at 5341
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1256
  

BBa_K5531010 (pET-Dual-HisRh3C-P15VP4-P4H)

Construction Design

This targeted gene, Rh3C-P15VP4 (BBa_K5531000), synthesized by a biotech company, contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15. Meanwhile, P4H (BBa_K5531012) is also co-expressed in this plasmid due to the lack of proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)) in procaryotes, which is essential for collagen stability. The pET-Dual-N-His-TEV (BBa_K5531006) serves as the vector. The homologous recombination method was employed to construct pET-Dual-HisRh3C-P15VP4-P4H (BBa_K5531010).

Fig. 1. Plasmid map of pET-Dual-HisRh3C-P15VP4-P4H

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 correctness toward the expected outcome (HisRh3C-P15VP4 is 2000 bp, and P4H is 755 bp). We selected colonies and sent them directly for sequencing. Figure 2 shows the success of pET-Dual-HisRh3C-P15VP4-P4H construction.

Fig. 2. The results of pET-Dual-HisRh3C-P15VP4-P4H

Characterization/Measurement

Later, the plasmid mounted with the gene of Rh3C-P15VP4 was transferred to E. coli DH5α to replicate. The extracted plasmid was transferred into E. coli BL21, which can help express His-Rh3C-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. After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein Rh3C-P15VP4 has a size of 85 kDa.

Fig. 3. The expression of His-Rh3C-P15VP4 using E. coli BL21.

Collagen-VP4 A was diluted to a concentration of 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 4, the samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation. Based on the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 4. The peaks for collagen-VP4 A ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimer and monomer macromolecules.

Fig 4. Native-PAGE analysis (left)of oligomeric states of collagen A; Size exclusion chromatographic (SEC) analysis(right) of oligomeric states of collagen A.

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.