Part:BBa_K3187000
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, 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 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 followed by a Western blotwas performed.
Assembly
The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation and are visualized with 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.
Figure 1: Western blot of all produced and purified proteins.
Fig. 1 shows that the band of the CP-LPETGG is approximatley found by the 49 kDa band. Consequently, the successful protein production was proven.CP-LPETGG is detected with Strep-Tactin-HRP.
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.
Figure 2: Ultracentrifugation of assembled VLPs
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).
Figure 3: Assembly of only coat proteins with LPETGG.
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 sfGFP.
Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and CP-LPETGG can be modified for this process
References
- ↑ 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]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1491
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |