Difference between revisions of "Part:BBa K4389001"
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===Biology===
 
===Biology===
 
The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of the Vaccinia virus [1] (Figure 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 [2]. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of the B5 protein that we derived using AlphaFold2 software (Figure 2). There are 4 Sushi domains that are also called Complement control protein (CCP) modules or short consensus repeats (SCR) [3]. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages [4].
 
The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of the Vaccinia virus [1] (Figure 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 [2]. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of the B5 protein that we derived using AlphaFold2 software (Figure 2). There are 4 Sushi domains that are also called Complement control protein (CCP) modules or short consensus repeats (SCR) [3]. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages [4].
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===Usage===
 
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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. The 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.
 
  
 
https://static.igem.org/mediawiki/parts/c/c8/1sushi.gif
 
https://static.igem.org/mediawiki/parts/c/c8/1sushi.gif
  
 
<strong>Figure 1.</strong> 3D model of 1st sushi domain of B5R protein  
 
<strong>Figure 1.</strong> 3D model of 1st sushi domain of B5R protein  
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 +
===Usage===
 +
 +
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. The 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
 
<!-- Add more about the biology of this part here

Revision as of 18:46, 8 October 2022


B5R 1st sushi

B5R 1st sushi


Biology

The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of the Vaccinia virus [1] (Figure 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 [2]. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of the B5 protein that we derived using AlphaFold2 software (Figure 2). There are 4 Sushi domains that are also called Complement control protein (CCP) modules or short consensus repeats (SCR) [3]. Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages [4].

1sushi.gif

Figure 1. 3D model of 1st sushi domain of B5R protein

Usage

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. The 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]