Part:BBa_K3187017
P22 Bacteriophage Coat Protein
Profile
| Name | Coat protein |
| Base pairs | 1293 |
| Molecular weigth | 46.9 kDa |
| Origin> | Bacteriophage P22 |
| Parts | Basic part |
| Properties | Assembly with scaffold proteins to VLPs |
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. 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 °C, 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 °C. 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]
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] We focused on the viral and bacteriophagic coat proteins, which are parts of their respective organisms’ capsid. The genetic information (DNA or RNA) is wrapped and protected by the capsid. During cell infection, the phage or virus transfer the genetic information into the infected cell. [8] Because of the high variety of coat proteins, we are focussing on one specific coat protein (BBa_K3187017), which is naturally found in the bacteriophage P22. This coat protein (CP) consists of 431 amino acids and its molecular weight is 46.9 kDa. Because of its significance as a part of the capsid, it represents one main part of our Virus‑like particle. Together with the scaffold protein (BBa_K3187021), they assemble to a VLP [9] and form the basis for our modular platform.
Methods
The basic part coat protein is produced and purified as the composite part (BBa_K3187000) (CP‑LPETGG) and (BBa_K3187001) (CP). For gene expression and protein purification as CP‑LPETGG the coding sequence contains a coat protein (BBa_K3187017) a LPETGG (BBa_K3187019), a T7 promoter, lac-operator and RBS (BBa_K3187029), a Short Linker 5AA (BBa_K3187030) T7 terminator (BBa_K3187032), and Strep-tagII (BBa_K3187025). The composite part coat protein without LPETGG (BBa_K3187001) contains a T7 promoter, lac-operator and RBS (BBa_K3187029), two terminators (T7Te terminator and rrnB T1 terminator (BBa_K3187036)), a Short Linker 5AA (BBa_K3187030) and Strep-tagII(BBa_K3187025). The production is performed in the E. coli strain BL21 (DE3) and it is purified with GE Healthcare ÄTKA Pure machine which is a machine for FPLC. To verify the successful production, a western blot is carried out.
Methods
The basic part coat portein was expressed, produced and purified as the composite part BBa_K3187000 (coat protein with LPETGG) BBa_K3187001 (coat protein). The production is performed in E. coli BL21 and it is purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. To verify the right production, a western blot was made.
Results
Figure 1: western blot of all produced and purified proteins.
Fig. 1 shows that the band of the CP‑LPETGG is approximately found by the 49 kDa band and the band of CP by 46.9 kDa. Consequently, the successful production was proven. CP‑LPETGG and CP were detected with Strep‑Tactin‑HRP.
TEM
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 .
We showed by dynamic light scattering (DLS) analysis (Fig. 5) that capsids containing only CP are smaller than P22‑VLPs containing both CP and SP. This was also confirmed by measuring VLPs and CP-only capsids in TEM images using ImageJ. Capsids which are only composed of CP measured average diameter of 53 nm ± 4.3 nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57 nm ± 3 nm (n=20; p < 0.005). What also became clear is that the presence of the LPETGG tag does not affect the size of the assembled CP-only capsid.
Want to know more about what we did with CP-LPETGG? Please visit the registry page of BBa_K3187000.
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]
- ↑ 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. [9]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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