Composite

Part:BBa_K3187001

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)


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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1458
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1504


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.

The assembled P22 VLPs occur as a procapsid. 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]


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 2: Diagram of DLS measurment of VLPs .

We showed by dynamic light scattering (DLS) analysis (Fig. 2) 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.

References

  1. Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
  2. Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
  3. 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]
  4. Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69-79 [4]
  5. 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]
  6. 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]
  7. Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [7]
  8. Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, 80- 88 [8]
  9. Roldão A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM, Virus-like particles in vaccine development., Expert Rev Vaccines, 2010, 9: 1149-1176 [9]
  10. 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]
  11. 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]





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