Difference between revisions of "Part:BBa K3187000"

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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 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. ^[1] 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).
The CP is originally found in the bacteriophage P22 and forms its capsid with the scaffold protein (SP) (BBa_K3187021). Heterologously expressed, coat proteins and scaffold poroteins assemble to a Virus-like particles (VLP). ^[2]

Of course there are more parts necessary in order to express the CP-LPETGG heterologously in E. coli BL21. As a backbone, the pET24 plasmid 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 the mCherry and CP-LPETGG. The linkage was verified with a SDS-PAGE. To identify whether the sortase produce multimers of coat proteins with LPETGG tag, CP-LPETGG and sortase were incubated for 3 h at 37 °C. It was verified via a 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 are collected with ultracentrifugation and are visualized with Transmission electron microscopy (TEM). The diameter of VLPs was measured with dynamic light scattering (DLS) analysis. For more information look at our wiki.

Results

Cloning and Expression

The successful cloning was proven with sanger sequencing and production with a western blot.

Fig. 1 shows that the band of the CP-LPETGG is approximatley found by the 49 kDa band. Consequently, the successful production was proven. CP-LPETGG was detected with Strep-Tactin-HRP.

Forming multimers

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

Fig. 2 shows the bands of BSA approximatley at 66 kDa and the CP-LPETGG at 49 kDa. When the proteins were combined, there are two bands, 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.

The SDS-PAGE shows multiple bands (Fig. 3), which relate to a higher molecular weight than mCherry or CP-LPETGG itself has. 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 alook 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. ^{[3] [4]} and the P22 Coat Protein accommodates a cysteine residue.

Assembly

The images of ultracentrifugation displays 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 reatment, 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.

The images of TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. As a result, the CPs produced using this part are fully functional. The CPs assemble with SPs and they can be modified on the surface (Fig. 4). Moreover, CPs also assemble without SPs (Fig. 3).

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

Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and 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.

As shown in the diagram, VLPs which only consist of coat proteins with LPETGG tag and VLPs made out of coat proteins without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). VLPs with both proteins have a diameter about 112 nm. Consequently, the LPETGG tag does not disturb the assembly of coat proteins.

References

1. ↑ 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 [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. ↑ 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, the Journal of biological chemistry, 2014, 289, 627-6638 [3]
4. ↑ 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 [4]