Difference between revisions of "Part:BBa K3187001"

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Usage and Biology

This part encodes the coat protein (CP) (BBa_K3187017) of the bacteriophage P22 capsid. Importantly, it must not be confused with coat proteins in membrane transport of eukaryotic cells. Coat protein is an umbrella term for many different proteins, which simplify the transfer of molecules between different compartments that are surrounded by a membrane. ^[1]
In the natural context of P22, its genetic information is included and protected by the capsid, before it is transferred into the host organism during infection. ^[2] Bacteriophagic coat proteins have been used for many purposes, for example vaccines ^[3] or drug delivery. ^[4]
The P22 coat protein (BBa_K3187001) consists of 431 amino acids and its molecular weight is 46.9 kDa. Because it is found in the structural components of viral proteins, it is an important part of Virus-like particles (VLP) as well. Together with the scaffold protein (BBa_K3187021), the proteins assemble to a VLP ^[5] and build the basis for our modular platform.

The coat proteins (BBa_K3187001) are heterologously expressed in E. coli BL21 (DE3). As backbone the pACYCT2 plasmid is used, containing a T7 promoter, lac-operator and RBS (BBa_K3187029). Moreover the part comprises a C-terminal Strep-tagII (BBa_K3187025), a Short Linker (5AA) (BBa_K3187030) and two terminators, T7Te terminator and rrnb T1 terminator (BBa_K3187036).

Methods

Cloning

The sequence of the coat protein ordered from Integrated DNA Technologies (IDT) was inserted in the pACYCT2 backbone. For this purpose, the Gibson asssembly was used. The sequence was verified by sanger sequencing through Microsynth Seqlab.

Purification

The heterologous expressed coat protein in E. coli was purified using a GE Healthcare ÄKTA Pure machine which is a machine for FPLC.

SDS-PAGE and western blot

To verify that the coat protein was heterologous produced, a SDS-PAGE followed by a western blot was performed.

Results

Cloning and Expression

The ordered sequence from IDT was cloned into the pACYCT2 plasmid with Gibson assembly and heterologous expressed in E. coli. The accuracy of cloning was controlled via sanger sequencing (Microsynth Seqlab ) and the production was observed using an SDS-PAGE and western blot.

Figure 1: western blot of all produced and purified proteins.

Fig. 1 shows that the CPs were detected with Strep-Tactin-HRP. The western blot shows a band corresponding to the size of approximately 46.9 kDa. So, the successful production was proven.

References

1. ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [1]
2. ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, 80- 88 [2]
3. ↑ Roldão A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM, Virus-like particles in vaccine development., Expert Rev Vaccines, 2010, 9: 1149-1176 [3]
4. ↑ Rohovie, Marcus J., Maya Nagasawa, and James R. Swartz. "Virus‐like particles: Next‐generation nanoparticles for targeted therapeutic delivery." Bioengineering & translational medicine 2.1 (2017): 43-57 [4]
5. ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [5]