Difference between revisions of "Part:BBa K4389000"
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===Usage and Biology=== | ===Usage and Biology=== | ||
The B5R gene encodes 42-kDa glycosylated type I membrane protein of envelope of Vacinia virus [1]. The protein B5R is highly conserved among multiple strains of vaccinia virus as well as in other orthopoxviruses, expanding the range of use for the detector of this protein. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of B5 protein that we derived using AlphaFold2 software. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages []. The recombinant protein must be refolded, as we did for other Vaccinia viral proteins, since inclusion bodies are unavoidably obtained. According to published protocols, all three proteins were expressed in E. coli, hence we decided to follow a similar procedure. PCR primer is designed and ordered for the amplification of the desired B5 sequence from the respective pVax1 constructs (Fig. 2), and specific restriction sites that are compatible with the pET23a vector (Fig.3) (NdeI at the 5’ end and XhoI at the 3’ end) will be added. Readily available pET-23a plasmid will be utilized for E.coli overexpression since it possesses a C-terminal 6His-tag, optimal for this cloning and further purification. <!-- Add more about the biology of this part here | The B5R gene encodes 42-kDa glycosylated type I membrane protein of envelope of Vacinia virus [1]. The protein B5R is highly conserved among multiple strains of vaccinia virus as well as in other orthopoxviruses, expanding the range of use for the detector of this protein. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of B5 protein that we derived using AlphaFold2 software. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages []. The recombinant protein must be refolded, as we did for other Vaccinia viral proteins, since inclusion bodies are unavoidably obtained. According to published protocols, all three proteins were expressed in E. coli, hence we decided to follow a similar procedure. PCR primer is designed and ordered for the amplification of the desired B5 sequence from the respective pVax1 constructs (Fig. 2), and specific restriction sites that are compatible with the pET23a vector (Fig.3) (NdeI at the 5’ end and XhoI at the 3’ end) will be added. Readily available pET-23a plasmid will be utilized for E.coli overexpression since it possesses a C-terminal 6His-tag, optimal for this cloning and further purification. <!-- Add more about the biology of this part here | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 16:07, 3 October 2022
B5R (all 4 sushi domains)
B5R
Usage and Biology
The B5R gene encodes 42-kDa glycosylated type I membrane protein of envelope of Vacinia virus [1]. The protein B5R is highly conserved among multiple strains of vaccinia virus as well as in other orthopoxviruses, expanding the range of use for the detector of this protein. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of B5 protein that we derived using AlphaFold2 software. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages []. The recombinant protein must be refolded, as we did for other Vaccinia viral proteins, since inclusion bodies are unavoidably obtained. According to published protocols, all three proteins were expressed in E. coli, hence we decided to follow a similar procedure. PCR primer is designed and ordered for the amplification of the desired B5 sequence from the respective pVax1 constructs (Fig. 2), and specific restriction sites that are compatible with the pET23a vector (Fig.3) (NdeI at the 5’ end and XhoI at the 3’ end) will be added. Readily available pET-23a plasmid will be utilized for E.coli overexpression since it possesses a C-terminal 6His-tag, optimal for this cloning and further purification. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 165
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]