Difference between revisions of "Part:BBa K4389000"

Revision as of 16:52, 4 October 2022

B5R (all 4 sushi domains)

B5R

Biology

The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of 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). Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages [3]. The recombinant protein must be refolded, as we did for other Vaccinia viral proteins since inclusion bodies are unavoidably obtained.

Figure 1. Intracellular mature (IMV) and extracellular enveloped (EEV) orthopoxvirus [4]

Figure 2. Modeled 3D structure of B5R protein

Usage

According to published protocols, B5R protein was expressed in E. coli, hence we decided to follow a similar procedure [5]. PCR primer is designed and ordered for the amplification of the desired B5 sequence from the respective pVax1 constructs (Figure 3), and specific restriction sites that are compatible with the pET23a vector (Figure 4) (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.

Figure 3. pVAX plasmid structure

Figure 4. pET plasmid structure

Part Funcionalization

Visualization

Gel electrophoresis (PCR products of 1st sushi and 4 sushi encoding DNA)

Figure 1. Results for gel electrophoresis of PCR products

Sequence and Features