Difference between revisions of "Part:BBa K4389000"

Latest revision as of 16:26, 11 October 2022

B5R (all 4 sushi domains)

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].

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

Figure 2. Modeled 3D structure of B5R protein by AlphaFold2 and animated by PyMol

Usage

Plasmid design

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

Gel electrophoresis

Sequence encoding all 4 sushi domains of B5R protein underwent PCR amplification and purification prior to being tested in gel electrophoresis. Gel electrophoresis results show the size of the gene appearing just below 750 bp with the actual size of the gene being 685 bp. Therefore, PCR amplification of the gene was successful.

Figure 5. B: PCR-amplified and purified nucleic acid sequence encoding all 4 sushi domains of B5R protein

Sequencing and colony PCR

E. coli BL-21 cells were transformed with the amplified ligation products of double-digested pET23a plasmid and sequences encoding 4 sushi domains. Colony PCR products of pET23a ligated with a sequence encoding 4 sushi domains traveled less distance due to the presence of T7 primers and therefore appeared higher in the gel than in control runs. The colony PCR product of pET23a ligated with the sequence encoding 4 sushi domains obtained from one successful colony was sent to DNA sequencing analysis. 2 samples with 4 sushi domains were sent to DNA sequencing analysis based on the colony PCR results. The chromatogram showed evenly-spaced peaks with no or little baseline noise. According to sequence alignment, all the sample sequences have aligned with the original DNA sequence encoding 4 sushi domains. Therefore, cloning all 4 sushi domains was successful.

Figure 6. E. coli BL-21 cells transformed with ligation product pET23a plasmid and sequence encoding 4 sushi domains

Figure 7.. Sequence alignment with plasmid DNA for 1st sushi and all 4 sushi domains. Image created in SnapGene.

SDS-PAGE

From the SDS-PAGE analysis, the mass of all 4 sushi domains of B5 protein was found to be ~25 kDa. The protein, however, was not expressed. As for Western blot, the results indicate the absence of protein expression as well.

Figure 7. SDS-PAGE analysis of B5 1st sushi domain protein. A)19°C. Lane 1- 2h and 0.5 mM IPTG. Lane 2- 4h and 0.5 mM IPTG. Lane 3-6h and 0.5 mM IPTG. Lane 4- ON and 0.5 mM IPTG. Lane 5- absent, Lane 6- 2h and 1 mM IPTG. Lane 7- 4h and 1 mM IPTG. Lane 8-6h and 1 mM IPTG. Lane 9- ON and 1 mM IPTG. B) same lanes but at 30°C. C) same lanes but at 37°C, Lane 5- control

Reference

1. Herrera, E., Lorenzo, M. M., Blasco, R., & Isaacs, S. N. (1998). Functional analysis of vaccinia virus B5R protein: essential role in virus envelopment is independent of a large portion of the extracellular domain. Journal of virology, 72(1), 294–302. https://doi.org/10.1128/JVI.72.1.294-302.1998

2. Engelstad, M., & Smith, G. L. (1993). The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence. Virology, 194(2), 627–637. https://doi.org/10.1006/viro.1993.1302

3. Ichinose, A., Bottenus, R. E., & Davie, E. W. (1990). Structure of transglutaminases. The Journal of biological chemistry, 265(23), 13411–13414.

4. Reid, K. B., & Day, A. J. (1989). Structure-function relationships of the complement components. Immunology today, 10(6), 177–180. https://doi.org/10.1016/0167-5699(89)90317-4

Sequence and Features