1. Add a Basic Part, BBa_K5531000 (Rh3C-P15VP4)

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Name: Rh3C-P15VP4

Base Pairs: 2329 bp

Origin: Human Type III Collagen; synthesized

Properties: Rh3C-P15VP4 contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15. Recombinant humanized type III collagen (Rh3C) was found to have stronger cell adhesion properties, suggesting a more stable triple helix conformation. Since type III collagen is widely distributed in the human body, it is more difficult to generate an immune response in the human body and can effectively form trimers.[1]

2. Add a Basic Part, BBa_K5531001 (PPG-P15VP4)

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Name: PPG-P15VP4

Base Pairs: 1609 bp

Origin: Eukaryotes; synthesized

Properties: The fundamental architecture of collagen sequences consists of recurring tripeptide motifs. The prevalent pattern is Gly-X-Y, with Gly denoting 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.[2]

3. Add a Basic Part, BBa_K5531002 (DKG50-P15VP4)

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Name: DKG50-P15VP4

Base Pairs: 1896 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 (P15) with one of the heterotrimeric collagen-like motifs, and the fusion-expressed proteins were purified and mixed to form oligomers in vitro.[3,4]

4. Add a Basic Part, BBa_K5531003 (PKG50-P4VP4)

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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) with one of the heterotrimeric collagen-like motifs, and the fusion-expressed proteins were purified and mixed to form oligomers in vitro.[3,4]

5. Add a Basic Part, BBa_K5531004 (EPG50-P8VP4)

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Name: EPG50-P8VP4

Base Pairs: 1881 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 (P8) with one of the heterotrimeric collagen-like motifs, and the fusion-expressed proteins were purified and mixed to form oligomers in vitro.[3,4]

6. Add a Basic Part, BBa_K5531005 (pET-24a)

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Name: pET-24a

Base Pairs: 5291 bp

Origin: Escherichia coli; synthesized

Properties: Commonly used vectors for E. coli

Figure 1. Plasmid map of pET-24a

7. Add a Basic Part, BBa_K5531006 (pET-Dual-N-His-TEV)

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Name: pET-Dual-N-His-TEV

Base Pairs: 5413 bp

Origin: Escherichia coli; synthesized

Properties: A plasmid has been designed for the simultaneous and effective co-expression of two target proteins in Escherichia coli. One of the two target proteins can be engineered with a His-tag, followed by a TEV protease cleavage site. This plasmid features two multiple cloning sites (MCS), each preceded by a T7 promoter/lac operon and a ribosome binding site (RBS), ensuring efficient initiation of target protein expression upon IPTG induction.

Figure 2. Plasmid map of pET-Dual-N-His-TEV

8. Add a Basic Part, BBa_K5531012 (P4H)

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Name: P4H

Base Pairs: 738 bp

Origin: Arabidopsis thaliana (Mouse-ear cress); synthesized

Properties: This enzyme facilitates the post-translational conversion of proline residues to 4-hydroxyproline within -Xaa-Pro-Gly- motifs present in proline-rich peptide sequences found in plant glycoproteins and various other proteins. It exhibits a preference for hydroxylating proline residues occupying the second position within -Pro-Pro-Gly- triplets. Hydroxyproline residues play crucial roles in the structural composition of numerous plant cell wall glycoproteins, including extensions, hydroxyproline-rich glycoproteins, lectins, and arabinogalactan proteins. Additionally, it can catalyze the hydroxylation of collagen-like peptides and hypoxia-inducible transcription factor peptides..[5]

Figure 3. Protein structure diagram of P4H

Usage and Biology:

Humanized type III collagen (Rh3C, a type III collagen, a homotrimer) or model collagen sequences (POGn, where O represents hydroxyproline) were used as innovative trimeric motifs, synthesized through synthetic biology techniques. These two trimeric motifs serve as stabilizing domains and native proteins to increase the immune response of the VP4. Here, conjugated Rh3C- P15VP4 and PPG-P15VP4 allow novel VP4 antigen oligomers to be formed.

Further, we utilized to reported artificially designed proline-free heterotrimeric collagen-like protein motifs ((PKG)n, (DKG)n, (EPG)n), three different serological Vp4 proteins (P4, P8, P15) were fused to one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed in vitro to form oligomers. In this way, a multivalent-oligomeric rotavirus vaccine in E. coli is achievable.

Add a Composite Part, BBa_K5531007 (pET-24a-DKG50-P15VP4); BBa_K5531008 (pET-24a-PKG50-P4VP4); BBa_K5531009 (pET-24a-EPG50-P8VP4); BBa_K5531010 (pET-Dual-HisRh3C-P15VP4-P4H); BBa_K5531011 (pET-Dual-HisPPG-P15VP4-P4H)

A: Plasmid construction

Using PET-Dual and PET-24a as vector plasmids, we inserted PPG, P4H, and Rh3C into PET-Dual, while EPG50 with p8, PKG50 with p4, and DKG50 with p15 were integrated into PET-24a. Based on the homologous recombination technique, we incorporated restriction sites to primers and applied the same restriction enzymes to the plasmids; then, the PCR fragment and linearized vector can be linked via homologous recombinase.

Firstly, we constructed the pET-Dual-HisRh3CP15VP4-P4H plasmid, amplified the Rh3C-P15-26-476 and P4H fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual protein prokaryotic expression plasmid) by double digestion, and then, by homologous recombination, the Subsequently, by homologous recombination, the HisRh3CP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H fragment was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisRh3CP15VP4-P4H was finally obtained.

Secondly, we continued to construct the pET-Dual-HisPPGP15VP4-P4H plasmid, amplified the PPG-P15-26-476 and P4H fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual-Protein Prokaryotic Expression Plasmid) by double-enzymatic cleavage, and then, using homologous recombination, we inserted the HisRh3CP15VP4 fragment between Xba1 and Sal1, and the P4H fragment between Nde1 and Xho1. Subsequently, by homologous recombination, the HisPPGP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H fragment was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisPPGP15VP4-P4H was finally obtained.

Thirdly, we continued to construct pET-24a-DKG50-P15VP4 plasmid, amplified DKG50-P15-26-476-FLAG fragments by PCR and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous recombination, we inserted the DKG50-P15VP4 fragments between Xba1 and Xho1 and finally received the recombinant plasmid pET-24a-DKG50-P15VP4. Xho1 to obtain the recombinant plasmid pET-24a-DKG50-P15VP4.

Fourthly, we continued to construct pET-24a-PKG50-P4VP4 plasmid, amplified PKG50-P4-26-476-HA fragments by PCR and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous recombination, we inserted the PKG50-P4VP4 fragments between Xba1 and Xho1 and finally received the recombinant plasmid pET-24a-PKG50-P4VP4.

Finally, we constructed pET-24a-EPG50-P8VP4 plasmid, amplified EPG50-P8-26-476-c-Myc fragments by PCR, and obtained linear plasmid backbone pET-24a by double digestion. Then, using homologous recombination, we inserted the EPG50-P8VP4 fragments between Xba1 and Xho1 and finally received the recombinant plasmid pET-24a- EPG50-P8VP4.

Figure 4. Electrophoresis for the target genes PKG50, DKG50, PPG, EPG50, and P4H

Figure 4 shows that the fragment lengths align with the expected results, confirming the successful amplification of the target genes.. After electrophoresis, the bands that contain the target DNA produced the required DNA fragment by gel extraction. The DNA extracted from the gel was inserted into the plasmid pET24a or pET-Dual via homologous recombination.

Subsequently, the concatenated plasmids A-E were introduced into E. coli DH5α through transformation. In Figure 5A, successful growth of isolated colonies is depicted, with selected colonies undergoing verification. Figure 5B shows that the length of Rh3CP15VP4 is about 2000 bp, and P4H is about 750 bp. After extracting the plasmid from the positive colony, the sequencing results in Figure 5C showed that Rh3CP15VP4 and P4H do not contain any gene mutation. It proves that the plasmid pET-Dual-HisRh3CP15VP4-P4H was successfully constructed.

Figure 5. The culture plate for E. coli DH5α(A), verification of colonies(B), and the sequencing results of pET-Dual-HisRh3CP15VP4-P4H(C)

The plasmid pET-Dual-HisPPGP15VP4-P4H was successfully constructed the same way as pET-Dual-HisRh3CP15VP4-P4H via homologous recombination, as shown in Figure 6.

Figure 6. The culture plate for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-Dual-HisPPGP15VP4-P4H (C)

The plasmid pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, and pET-24a-EPG50-P8VP4 were successfully constructed using homologous recombination, as shown in Figure 7.

Figure 7. The culture plates for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4 (C)

B: Protein Expression and Purification

We first transformed the five plasmids into E.coli BL21 (DE3) to test the protein's expression. Figure 8A shows that the isolated colonies were successfully grown and selected for colony verification. After the E.coli BL21 had been cultured for 12-16h, single colones were selected and examined via PCR. Figure 8B shows the electrophoresis results after PCR.

Figure 8. The culture plates for E. coli BL21 (A), verification of colonies (B) results of pET-Dual-HisRh3CP15VP4-P4H, pET-Dual-HisPPGP15VP4-P4H, pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4

After the colonies (BL21) were verified, they were correctly inoculated into liquid LB medium and cultured until reaching an OD600 of 0.6. Inducer IPTG was then added to induce protein expression. Following overnight cultivation at 16°C, the cells were lysed, and the protein was purified for subsequent analysis via SDS-PAGE and Native-PAGE electrophoresis.

SDS-PAGE gels showing the purification results. Protein purification of the five proteins A-E was examined using nickel column affinity chromatography. As can be seen from the figure, there is a single band at the size of the target proteins. This further indicates that the proteins can be correctly folded and expressed after induction and are solubilized for expression.

Figure 9. The expression of His-Rh3CP15VP4 (A), His-PPGP15VP4 (B), DKG50-P15VP4 (C), PKG50-P4VP4 (D), EPG50-P8VP4 (E) using E. coli BL21. Tips: The sample order from left to right is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

C: Functional Analysis

Collagen-VP4 A, B, C, D, and E were fermented separately in 400 mL of inductive culture medium. The proteins were then purified using a one-step nickel affinity chromatography method, as shown in Figure 10. The yield rates for each collagen were 3.379, 2.557, 2.256, 3.723, and 1.085 mg/L, respectively. Collagen-VP4 A, B, and the C-D-E-VP4 complex (in a 1:1:1 ratio) were diluted to 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 10, 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.

Figure 10. Native-PAGE electrophoresis showing the protein trimer formed by Rh3CP15VP4(A) and PPGP15VP4(B). The heterotrimeric collagen is composed of DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E)

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 11. The peaks for collagen-VP4 A and B 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. 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. Additionally, we calculated the peak area ratio of trimer to monomer for the prominent peaks to assess the assembly efficiency of the collagens. The ratios were 1.27 for collagen-VP4 A, 1.24 for collagen-VP4 B, and 0.60 for the C-D-E VP4 complex/monomers.

Figure 11. Size exclusion chromatographic (SEC) analysis of homotrimeric collagen-VP4, Rh3CP15VP4(A), and PPGP15VP4(B), heterotrimeric C-D-E-VP4 complex. Tips: DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E)

We investigated the sequences of humanized type III collagen (Rh3) and model collagen (PPG) as novel trimer motifs, which were fused with rotavirus VP4 protein to create the VP4 antigen trimer. Prokaryotes lack prolyl 4-hydroxylases (P4H) enzymes necessary for collagen stability. In future studies, P4H derived from giant viruses will be co-expressed with the VP4 protein mentioned above to achieve the stability of VP4 oligomers. We used the reported artificially designed heterotrimer-like collagens ((PKG)n, (DKG)n, (EPG)n) to fusion three serological Vp4 proteins (P4, P8, P15) with one of the heterotrimer-like collagens. The oligomers were formed by mixing the purified protein in vitro. In this way, an oligomer rotavirus vaccine can be prepared for E. coli without P4H modification.

Our strategy involves developing a VLP vaccine comprising non-infectious particles lacking viral genetic material. Introducing VLPs into the body elicits an immune response. While several attenuated rotavirus vaccines are available, ongoing research and enhancements are required to optimize their safety and efficacy. Genetically engineered vaccines represent a promising approach for preventing rotavirus infections in a safer and more effective manner.

[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.

[2]     Saikumari Y K , Balaram P .An internally quenched fluorescent substrate for collagenase[J].Biopolymers, 2010, 90(2):131-137.

[3]     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.

[4]     Zezhong LIU, Jie ZHOU, Yun ZHU, Lu LU, Shibo JIANG. Applications of the recombinant human collagen type Ⅲ-based trimerization motif in the design of vaccines to fight against SARS-CoV-2 and influenza virus[J]. Synthetic Biology Journal, 2024, 5(2): 385-395.

[5]     Rutschmann C, Baumann S, Cabalzar J, Luther KB, Hennet T. Recombinant expression of hydroxylated human collagen in Escherichia coli. Appl Microbiol Biotechnol. 2014 May;98(10):4445-55.