Difference between revisions of "Part:BBa K4389001"

Latest revision as of 16:27, 11 October 2022

B5R 1st sushi

Biology

The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of the Vaccinia 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 [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. 3D model of 1st sushi domain of B5R protein

Usage

Plasmid modeling

Figure 4. pET plasmid structure with B5R (1st sushi)

Gel electrophoresis

The sequence encoding the 1st sushi domain 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 below 250 bp with the actual size of the gene being 187 bp. Therefore, PCR amplification of the gene was successful.

Figure 5. A: PCR-amplified and purified nucleic acid sequence encoding 1st sushi domain of B5R protein

Sequencing and PCR colony

E. coli BL-21 cells were transformed with the amplified ligation products of double-digested pET23a plasmid and sequences encoding 1st sushi domain. Colony PCR products of pET23a ligated with sequence encoding 1st sushi domain traveled less distance due to the presence of T7 primers, and therefore appeared higher in the gel than control runs. The colony PCR products of pET23a ligated with sequence encoding 1st sushi domain obtained from four successful colonies were sent to DNA sequencing analysis. 6 samples (reverse and forward for each colony) with 1st sushi domain were sent for 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 the 1st sushi domain. Therefore, cloning of 1st sushi domain was successful.

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

SDS-PAGE

From the SDS-PAGE analysis, the mass of the 1st sushi domain of B5 protein was found to be ~6 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 all 4 sushi domains 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. Lane 5- control C) same lanes but at 37°C.

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