Difference between revisions of "Part:BBa K3187000"

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, T7 terminator, 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 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 polyG recognition sequence is composed of four glycines (GGGG) (BBa_K3187018)
The CP is originally found in bacteriophage P22 and forms its capsid with the scaffold protein(SP) (BBa_K3187021). Heterologously expressed, CPs and SPs assemble to a Virus-like particle (VLP). ^[2]

Of course there are more parts necessary in order to express the CP-LPETGG heterologously in E. coli. As 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-tagII (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-tagII.

SDS-Page and Western blot

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

Sortase-mediated Ligation

In order to characterize CP-LPETGG, different assays are made. The possibility of modification is tested with mCherry and sortase as well as sfGFP and sortase . The sortase has linked the mCherry or sfGFP and CP-LPETGG. The linkage is verified with a SDS-Page. To identify whether the sortases produce multimers of coat proteins, 6 µL coat proteins and 3 µL sortase have been incubated for 3 h at 37°C. It was verified with a SDS-Page. For more information look at our wiki.

Assembly

The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation ultracentrifugatione and are visualized with TEM TEM. 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 cloning and expression was proven.CP-LPETGG is detected with Strep-Tactin-HRP.

Sortase-medited Reaction

The possibility of modification is verified with a SDS-Page which shows ….. warten auf Text von Sortase leuten BILD BIldunterschrift: SDS-Page of modified CP-LPETGG with mCherry/sfGFP

The results on the SDS-Page of testing sortase-mediated linkage between coat proteins with sortase suggested that Sortase A7M and Sortase A5M produce CP multimers, because wild type Sortase A is able to link two proteins together via disulfide bridges [1,2] and the P22 Coat Protein accommodates a cysteine residue. BILD

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. Transmission electron microscopy (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 scaffold proteins (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

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]