Coding

Part:BBa_K3187017

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)
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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 purified CP-LPETGG and CP.

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.

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.

Want to know more about what we did with CP-LPETGG? Please visit the registry page of BBa_K3187000.

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. ↑ 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]



Uses and Improvements

Description of image

Imperial-College 2024
This part is re-designed by Team Imperial_College in iGEM 2024, as part of their project to make compartmentalized artifical organelle that produces rubber. Their work also made this part purifiable through a much easier His-tag purification method.

This part encodes a His-tagged P22 Salmonella typhimurium bacteriophage virus capsid coat protein, adapted from BBa_K3187017. This part is reported to be translated to form a soluble virus-like-particle, that can encapsulate specifically proteins with a P22 scaffold protein(SP, BBa_K3187021) domain, for instance our part HRT2-SP (BBa_K5416001) [1][2].

This part is characterized in its composite format BBa_K5416062, with functions of:

  • Forming purifiable artifical organelle
  • Encapsulating enzymes
  • Producing hydrophibc internal environment
  • Producing small amount of rubber

Design

In this part, the the location of His-tag is carefully chosen to ensure the surface display of which on only the outter surface of the VLP. This is achieved via analysing the sturcture of P22 coat protein [1].

Fig 1: Structure of P22 coat protein in its VLP shell, 6 peptide chain symetically arranged to assembles a repeating unit from the entire VLP. The amino acid from N to C terminus of the unit is colored in blue to red.

Through analysing the structure of P22 coat protein, the C terminus of this virus shell is found to be exposed and pointing outward. Hence the 6xHis-tag is immediately placed at the last residue of the P22 coat protein.

Characterization

This part is characterized in composite format with HRT2-SP (BBa_K5416001) to form a enclosed environment for rubber production.

Expression

Fig 2: SDSPAGE analysis result of IPTG-induced E. coli cell (BL21(DE3)) expressing this part and HRT2-SP (BBa_K5416001), cultured 16hr at 25C. From left to right: lane L: Transgen 10-180kDa protein marker; lane 1: cell pellet after lysed with BugBuster with 0.2mg/ml lysozyme for 30min at room temperature; lane 2: lysate supernatant centrifuged at 3k rpm for 10min; lane 3: lysate supernatant centrifuged at 12k rpm for 10min; lane 4: cells sampled directly from the culture media; lane 5: 0.45um PDMV-filtered lysate (after centrifuged at 3k rpm); lane 6: first wash (with PBS) after protein loaded to Ni-NTA columns; lane 7: second wash (PBS with 2mM imidazole) of the Ni-NTA column; lane 8: 5ul of 20x concentrated elute (200mM imidazole); lane 9: 80x concentrated lysate supernatant, 5ul. All lanes except lanes 8 and 9 were loaded with 10ul of samples. The P22 capsid protein around 46kDa and HRT2trunc protein around 25kDa were identified with red arrows in the gel.

A clear evidence of the expression of P22-His is acquired through the SDS-PAGE analysis (band at 46kDa). Where it was also evidenced that this part is expressed in a highly soluable format. Through the process of protein purification, it is suggested that this part binds to the Ni-NTA columns firmly (no tracable amount in washes with PBS), and can be selectively eluted imidazole solutions. The presence of HRT2-SP in the eluted potion of the VLP also suggesting that a enclosed capsule is formed.Finally, the results above evidenced the design of this part enables the use of his-tag purification methods to acquire VLPs, which is more cost efficent than ultracentrifuging at over 100k rpm.

VLP Transmission Elelctron Microscopy

To futher investiage the structure of the VLP purified in this format, a TEM is conducted to image the euluted flowthrough.

Fig 3: Negatively stained transmission electron microscopy (TEM) imaged with 80kV at 15k magnification for the VLP concentrated in PBS. Spherical VLPs have been identified in the sample (with red arrows) of approximately 50nm in diameter. Regions are zoomed out on the left side of the image where the bar corresponds to 50nm.

The concentrated VLP after purification was sent to our external contractor (Service Bio, China), where the TEM was then pictured. The image X, shows several spots of spherical objects approximately 50nm in diameter exhibiting the characteristics to be our VLP [2].

BODIPY Staining

In the composite design, the VLP is produced to hold rubber molecules, hydrophobic aliphatic chains, inside an enclosed environment. To investigate if spacially locating the HRT2-SP (rubber synthase with scaffold protein) protein into our VLP would enable the formation of natrual rubber particles.

This hypothesis is studied using BODIPY staining technique, which this stain binds specifically to intracellular aliphatic compounds and has been thus used to stain lipid bodies and rubber particles in vivo [3][4]. In which we have compared the staining result of E. coli cells expressing this part with the wild-type stains (with empty backbone pET28a) and non-induced strains.

Fig 4: BODIPY staining of BL21 transformed with pET28a (pET) and the composite part (P22 + HRT2-SP), cells were induced with 0mM (-) and 1mM IPTG (+) for 16hrs at 25C, respectively. Each group is carried out with five repeats where the fluorescence of the cells was measured with excitation at 485nm and emission at 520nm (green) to quantify the BODIPY inside the cell. pET28a transformants serves as a global control.

After staining and washing off excessive BODIPY molecules with PBSG (5% glycerol), the fluorescence of the cells was measured with the plate reader, with a significant increase in the VLP expressing strain compared to the pET28a control observed. Where a student t-test revealed a p value smaller than 0.05. This indicates a presence of hydrophobic core inside the VLP, and yet not disrupting the structure of the capsule (which would otherwise leads to the fusion of hdyrophobic body to the cell membrane, reducing BODIPY signals).

References

1. Das, S., Zhao, L., Elofson, K. and Finn, M.G. (2020). Enzyme Stabilization by Virus-Like Particles. Biochemistry, 59(31), pp.2870–2881. doi:https://doi.org/10.1021/acs.biochem.0c00435.
2. Xiao H, Zhou J, Yang F, Liu Z, Song J, Chen W, Liu H, Cheng L. Assembly and capsid expansion mechanism of bacteriophage P22 revealed by high-resolution cryo-EM structures. Viruses. 2023 Jan 26;15(2):355.
3. Yokota S, Gotoh T. Effects of rubber elongation factor and small rubber particle protein from rubber-producing plants on lipid metabolism in Saccharomyces cerevisiae. Journal of bioscience and bioengineering. 2019 Nov 1;128(5):585-92.
4. Govender T, Ramanna L, Rawat I, Bux F. BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresource technology. 2012 Jun 1;114:507-11.

Index

MALNEGQIVT LAVDEIIETI SAITPMAQKA KKYTPPAASM QRSSNTIWMP VEQESPTQEG WDLTDKATGL LELNVAVNMG EPDNDFFQLR ADDLRDETAY RRRIQSAARK LANNVELKVA NMAAEMGSLV ITSPDAIGTN TADAWNFVAD AEEIMFSREL NRDMGTSYFF NPQDYKKAGY DLTKRDIFGR IPEEAYRDGT IQRQVAGFDD VLRSPKLPVL TKSTATGITV SGAQSFKPVA WQLDNDGNKV NVDNRFATVT LSATTGMKRG DKISFAGVKF LGQMAKNVLA QDATFSVVRV VDGTHVEITP KPVALDDVSL SPEQRAYANV NTSLADAMAV NILNVKDART NVFWADDAIR IVSQPIPANH ELFAGMKTTS FSIPDVGLNG IFATQGDIST LSGLCRIALW YGVNATRPEA IGVGLPGQTA HHHHH H

The amino acid sequence of this improved part is dispalyed here, to ease the process of codon optimization of protein engineering. The sequence in dark purple (html #993366) encodes the wild-type P22 coat protein, where the 6xHis-tag is colored in pink (#ff99cc).

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None