Part:BBa_K3187001
P22 Bacteriophage Coat Protein expression cassette
Profile
| Name | Coat protein |
| Base pairs | 1293 |
| Molecular weight | 46.9 kDa |
| Origin | Bacteriophage P22 |
| Parts | Coat protein, T7 promoter, lac-operator, RBS, T7Te terminator, rrnB T1 terminator, Short Linker 5AA, Strep-tagII |
| Properties | Assembly with scaffold protein to a Virus-like particle |
Usage and Biology
The P22 VLP originates from the temperate bacteriophage P22. Its natural host is Salmonella typhimurium.
Since it was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for temperate phages.
To date, nearly everything is known about its lifecycle including virions. Because of that and its specific properties it generates
an accessible VLP platform.
[1]
An assembled P22 VLP consists of 420 copies of coat protein (CP: BBa_K3187017) and 100 to 300 copies of scaffold
protein (SP: BBa_K3187021 ).
[2]
The shell of the VLP is formed by the 46.6 kDa CP. The coat protein occurs in one configuration, which contains a globular
structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric units, which form
the icosahedral structure of the VLP.
[3]
The 18 kDa SP is required for an efficient assembly and naturally consists of 303 amino acids. It has been shown, that an
N-terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D-structure is composed of segmented helical
domains, with little or no globular core. In solution is a mixture of monomers and dimers present.
[4]
When purified CPs and SPs are mixed, they self-assemble into VLPs.
P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60 degrees, the CP rearranges, forming the expanded shell form (EX). This form has a diameter of about 58 nm and the volume is doubled compared to the one of the procapsid. The expanded shell form changes into the whiffleball form (WB) when heated further up to 70 degrees. The whiffleball has 10 nm pores, while the procapsid or the expanded shell form only have 2 nm pores. [5] Furthermore, the P22 VLP consists of SP and CP, but it also can assemble with only CPs. If it assembles without SP it can form two sizes of capsids. The small capsid is built as a T = 4 icosahedral lattice with a diameter between 195 Å and 240 Å. The larger capsid also has an icosahedral lattice, but it is formed as T = 7. T being the "triangulation number", a measure for capsid size and complexity. Moreover, it is like the wild type VLP, which includes the SP. The diameter of the wild type VLP, is between 260 Å and 306 Å. Each capsid consists of a 85 Å thick icosahedral shell made of CP. [6]
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.
[7]
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.
[8]
Bacteriophagic coat proteins have been used for many purposes, for example vaccines
[9]
or drug delivery.
[10]
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
[11]
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.
Assembly
A VLP is assembled only with coat proteins without a tag and concentrated by ultracentrifugation. To verify the assembly, the VLPs were detected by transmission electron microscopy (TEM). The hydrodynamic diameter was measured with dynamic light scattering analysis (DLS).
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.
The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis.
Figure 5: Diagram of DLS measurment of VLPs .
References
- ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
- ↑ Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions,Nature Structural Biology, 2003, 10: 131-135 [3]
- ↑ Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69-79 [4]
- ↑ Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 [5]
- ↑ P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.,Biophysical Journal, 1998, 74: 559-568 [6]
- ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [7]
- ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, 80- 88 [8]
- ↑ Roldão A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM, Virus-like particles in vaccine development., Expert Rev Vaccines, 2010, 9: 1149-1176 [9]
- ↑ 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 [10]
- ↑ 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. [11]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1458
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1504
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