Part:BBa_K5416061

P22-His (His-tagged P22 Salmonella Phage Capsid Coat Protein)

Imperial-College 2024

This part is designed by Team Imperial_College in iGEM 2024. It is reported to form an artificial organelle which serves as an enclosed chamber for natrual rubber production.

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.<b>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 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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 532
  Illegal AgeI site found at 933
  
• 1000
  COMPATIBLE WITH RFC[1000]