Composite

Part:BBa_K3187000

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)
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P22 Bacteriophage Coat Protein with LPETGG Tag for Sortase-mediated Ligation


Profile

Name Coat protein with LPETGG in pET24
Base pairs 1359
Molecular weight 49.0 kDa
Origin Synthetic
Parts Coat protein, LPETGG, T7 promoter, lac-operator, RBS, T7 terminator, Short Linker 5AA, Strep-tagII
Properties Assembly with scaffold proteins to VLPs which can be modified exterior.

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 gene 5 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] If purified CPs and SPs are mixed, they 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 wiffleball form (WB) when heated further up to 70 degrees. The wiffleball 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 only with 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. 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 out of CP. [6]

The coat protein with LPETGG (CP-LPETGG BBa_K3187000) consists of 452 amino acids, which are encoded by 1359 DNA base pairs. The whole protein has a mass of 49.0 kDa. Its relevant parts are the coat protein (CP) (BBa_K3187017) and the LPETGG sequence (BBa_K3187019).
LPETGG is a synthetic sequence that is recognized by the enzyme family Sortase A and allows the coupling of CP with other peptides and proteins. For this, the sortase cleaves between the amino acids threonine (T) and glycine (G), and threonine forms an amide bond with another polyG sequence. [7] We used the Sortase A7M (BBa_K3187028) and Sortase A5M (BBa_K3187016). The used polyG recognition sequence is composed of four glycines (GGGG) (BBa_K3187018) [8]

Of course there are more parts necessary in order to express the CP‑LPETGG heterologously in E. coli BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is transcribed into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the protein. The T7 promoter (BBa_K3187029) is recognized by the T7 polymerase. In order to regulate the protein production, the lac-operator (BBa_K3187029) was used. Furthermore, a RBS (BBa_K3187029) is in the construct and a Short Linker (5AA) (BBa_K3187030) is found between CP and LPETGG. The T7 terminator (BBa_K3187032) and Strep-tag II (BBa_K3187025) are located downstream of the coat protein CDS.

Methods

Cloning

The CP-LPETGG was cloned into the pET24-backbone with restriction and ligation . To do this, the CP‑LPETGG, as well as the T7 promoter and the lac-operator sequence, was ordered from Integrated DNA Technologies (IDT). To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The CP-LPETGG was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-tag II.

SDS-PAGE and western blot

To verify that the CP-LPETGG was produced, a SDS-PAGE followed by a western blot was performed.

Forming multimers

To test whether coat proteins with a LPETGG tag form stable multimers, the concentration of CP‑LPETGG was increased in the presence of the protein BSA. The concentrated protein solution was heated up to 95 °C and a SDS-PAGE was performed to verify the stability of multimers.

Sortase-mediated Ligation

In order to characterize CP‑LPETGG, different assays were performed. The possibility of modifying the CP was tested with mCherry and Sortase A7M. The Sortase A7M successfully linked mCherry and CP-LPETGG. The linkage was verified with a SDS‑PAGE. To identify whether the Sortase A7M or Sortase A5M produce multimers of coat proteins with LPETGG-tag, CP-LPETGG and Sortase A7M and Sortase A5M were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS‑PAGE. For more information, please have a look at our wiki.

Assembly

The assembly was tested in vivo and in vitro. The assembled VLPs were collected with ultracentrifugation and were visualized with transmission electron microscopy (TEM). Therefore, the in vivo assembled VLPs are purified with size-exclusion chromatography (SEC) (Sephadex-100 column) [3] The diameter of VLPs was measured with dynamic light scattering (DLS) analysis.

Results

Cloning and Expression

The successful cloning was confirmed with sanger sequencing and successful production of the VLPs was confirmed with a western blot.

Figure 1: Western blot of all produced and purified proteins.

Fig. 1 shows that the band of the CP-LPETGG is can be seen at approximately 49 kDa. Consequently, the successful production was proven. CP-LPETGG was detected with Strep-Tactin-HRP.

Forming multimers

The SDS‑PAGE (Fig. 2) suggests that the CP-LPETGG does not form multimers , even if it is in high concentration.

Figure 2: SDS-PAGE of BSA and CP-LPETGG.

Fig. 2 shows the bands of BSA at approximately 66 kDa and the CP-LPETGG at approximately 49 kDa. When the proteins were combined, two bands can be seen, one of CP-LPETGG and one of BSA. Hence, no multimers were formed.

Sortase-medited Reaction

The possibility of modification was shown with a SDS-PAGE, which shows GGGG-mCherries linked to several CP-LPETGG.

Figure 3: SDS-PAGE of CP-LPETGG modified with mCherry by Sortase A7M and Sortase A5M.

The SDS-PAGE shows multiple bands (Fig. 3), which relate to a higher molecular weight than mCherry or CP-LPETGG themselves have. The bands located between 55 kDa and 70 kDa most likley show the linked CP-LPETGG and GGGG-mCherry, as we expected the product to be approximately XY kDa. Want to know more about how the modification works? Please have a look at our wiki.

The results on the SDS-PAGE of testing sortase-mediated linkage between coat proteins with LPETGG-tag with Sortase A7M and Sortase A5M (Fig. 2) suggested that Sortase A7M and Sortase A5M produce CP-LPETGG multimers, because wild type Sortase A is able to link two proteins together via disulfide bridges [9] [10] and the P22 Coat Protein accommodates a cysteine residue.

Figure 2: SDS-PAGE of sortase-mediated linkage between several coat proteins with LPETGG-tag.

Assembly

Ultracentrifugation was used to harvest VLPs after in vivo and in vitro assembly

in vivo assembled VLPs

For extracting the VLPs, which consits of SP and with sfGFP modified CP-LPETGG, directly from cell broth we first lysed the cells by sonication and got rid of debris by two centrifugation steps at 12,000 x g. Afterwards ultracentrifugation with a sucrose cushion (35% w/v) at 150,000 x g was used as a first concentration step. The resulting sediment contained fluorescent material which we suspected to contain a concentrated fraction of VLPs.

Figure 3: Cell broth after ultracentrifugation. Supernatant containing sfGFP-SP and CP while VLPs collected in the sediment

Ultracentrifugation sediment most likely still contains monomeric proteins and small amounts of cell debris that can be harmful in some applications due to high endotoxin levels. For getting rid of these contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex-100 column) [3] . After SEC the elution sample with the highest suspected VLP concentration (based on UV absorption) was imaged with transmission electron microscopy (TEM). Numerous capsids in the correct size range were clearly visible. This lead us to believe that ultracentrifugation, as well as SEC treatment, do not interfere with capsid integrity while separating VLPs from other contaminants. Chromatography dilutes the sample significantly which is not optimal for analytic purposes. This is why a second ultracentrifugation treatment would be required for re-concentration of purified capsids as [3] suggest.

Figure 4: Intact P22-VLPs after size exclusion chromatography

in vitro assembled VLPs

The images of ultracentrifugation show that monomeric proteins were separated from assembled capsids by ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation treatment, a sediment was clearly visible in the centrifuge tube, which we suspected to mainly contain VLPs. TEM was used to image capsids taken from the sediment. For increased contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually intact VLPs all over the sample, measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly, visit our wiki.

Figure 2: Ultracentrifugation of in vitro assembled VLPs

The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, the CPs produced using this part must be fully functional. The CPs assemble with SPs and can be modified on the surface (Fig. 4). Moreover, CPs also assemble without SPs (Fig. = 3).

Figure 3: Assembly of only coat proteins with a LPETGG-tag.

Fig. 3 shows that no scaffold proteins are necessary for assembly.

Figure 4: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.

Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.

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 53nm±4.3nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57nm±3nm (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. ↑ Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman, Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner, plos one, 19.02.2014 [7]
  8. ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [8]
  9. ↑ Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, Craik DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A, Journal of biological chemistry, 2014, 289, 627-6638 [9]
  10. ↑ Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, Chenggang Wu, Neda Jooya, Andrzej Joachimiak, Asis Das, Hung Ton-That‡2, A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 [10]
  11. ↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [11]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1491
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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