Part:BBa_K3187003

Superfolder Green Fluorescence Protein x P22 Bacteriophage Scaffolding Protein Fusion

Profile

Name	sfGFP-scaffol protein-fusion protein
Base pairs	1547
Molecular weight	46.1 kDa
Origin	Enterobacteria phage P22; Aequorea victoria
Parts	T7-Promoter, lac-operator, RBS(g10 leader sequence), sfGFP, Strep-tag II, scaffold protein (SP), double terminator (rrnB T1 terminator and T7Te terminator)
Properties	P22 capsid assembly; loading the capsid with sfGFP

Methods

Cloning

The fusion protein was cloned into the pACYC2 backbone with Gibson Assembly . To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The protein was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA 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.

Assembly

The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation ultracentrifugatione 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 SP has a molecular weight of approximatley 45 kDa. This is about the expected size of 46.1 kDa. Two additional bands in this lane can be observed. One at est. 25 kDa and one between 25 and 37 kDa. The lower band may be sfGFP and upper band scaffold protein. We came to this conclusion by comparing the lane of Sp-sfGFP with lanes of only SP and with sfGFP with TEV cleavage site. Those two bands are probably produced by the denaturing of the SP-sfGFP fusion protein. During denaturation for SDS-PAGE sample preparation, the fusion protein can break in two parts, sometimes it breaks in front and sometimes after the Strep-tag. This is indicated by the fact that both bands are stained by an anti strep-tag western blot. The band of Strep-tag and SP can be observed at a size of est. 30 kDa. This is larger than the expected, theoretical size of SP at about 18 kDa. Because the plasmid used for expression was verified by sequencing before, and the fusion protein has the right size when it is not broken from sample preparation, we suspect that the protein is the right one and it just behaves unexpected in this SDS PAGE.

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 sGFP.

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

References

Sequence and Features BBa_K3187003 SequenceAndFeatures