Part:BBa_K5531009

pET-24a-EPG50-P8VP4

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 5062
  Illegal NotI site found at 5120
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4957
  Illegal BamHI site found at 5095
  Illegal XhoI site found at 5129
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 3177
  Illegal NgoMIV site found at 3337
  Illegal NgoMIV site found at 4925
  
• 1000
  COMPATIBLE WITH RFC[1000]

BBa_K5531009 (pET-24a-EPG50-P8VP4)

Construction Design

Using the backbone of pET-24a (BBa_K5531005), proline-free heterotrimeric collagen-like protein motif EPG50 is fused with serological P8-VP4 via chemical synthesis. The homologous recombination method was employed for the construction of pET24a-EPG50-P8VP4 (BBa_K5531009).

Fig. 1. Plasmid map of pET-24a-EPG50-P8VP4

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 correctness toward the expected outcome (EPG50-P8VP4 is 2000 bp); we selected colonies and sent them directly for sequencing. Figure 2 shows the success of pET24a-EPG50-P8VP4 construction.

Fig. 2. The results of pET24a-EPG50-P8VP4

Characterization/Measurement

Later, the plasmid pET-24a-EPG50-P8VP4 was transferred to E. coli DH5α to replicate. The extracted plasmid was transferred into E. coli BL21, which can help express His-EPG50-P8VP4. 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. The target protein EPG50-P8VP4 has a size of 67.5 kDa.

Fig. 3. The expression of EPG50-P8VP4 using E. coli BL21.

Characterization of Collagen Oligomers

Collagen of the C-D-E-VP4 complex (in a 1:1:1 ratio) 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 the C-D-E-VP4 complex also ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.57 CV, 0.68 CV, 0.72 CV, and 0.77 CV, indicating a trimer at 0.57 CV and the other peaks corresponding to the monomers of C, D, and E.

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

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.