Difference between revisions of "Part:BBa K3187004"

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	The successful cloning was proven with Sanger sequencing and production with a western blot.		The successful cloning was proven with Sanger sequencing and production with a western blot.
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Revision as of 12:02, 21 October 2019

TEV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X Superfolder Green Fluorescence Protein

Profile

Name	TEV site-polyG-sfGFP
Base pairs	1028
Molecular weight	27.8 kDa
Origin	Tabacco Etch Virus (TEV); Aequorea victoria
Parts	T7-Promoter, lac-operator, RBS (g10 leader sequence), TEV protease recognition sequence, polyG-tag, sfGFP, Strep-tag II, T7 terminator
Properties	After cleavage by the TEV protease, the polyG tag can be used to fuse sfGFP to the Sortase A recognition sequence(LPTEGG)

Usage and Biology

The TEV protease is cleaving a protein after a specific sequence between Glutamine and Serine or Glycine ^{[1] [2]} . We are using this to create a free N-terminal polyG sequence in front of sfGFP so we can use it as substrate in a Sortase A mediated reaction ^{[3] [4] [5]} .
sfGFP is a variant of the fluorescence protein GFP that was originally isolated from the jellyfish Aequorea victoria. It has a short maturing time of 13.6 min, has an extinction maximum at 485 nm and an emission maximum at 510 nm. ^{[6] [7]}
At the end of the sfGFP a strep tag was added to enable easy protein purification.
The part contains a T7 promoter so it can be transcribed by T7 polymerase, and a lac operator so protein expression can be induced by IPTG.

Methods

Cloning

The TEV-site was added to sfGFP through Gibson overhang primers and cloned into the pET24 via 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 TEV-site with sfGFP was produced, a SDS-PAGE followed by a western blot was performed.

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 sfGFP with TEV cleavage site has a molecular weight of less than 25 kDa. The expected weight is 27.8 kDa.

Sortase-mediated Ligation of the GGGG-tagged sfGFP

The protein was cleaved with TEV protease to ptoduce the N-terminal polyG tag. The cleaved protein was then used in different characterisation assays of different Sortase variants.

VLP modification

The cleaved protein was further used for modification of assembled virus-like particles.

In order to demonstrate the integrity of our modified VLPs we used capsids from the same sample for DLS and electron microscopy which confirms the presence of intact VLPs. The size distribution shows that they still pose a monodisperse species, even though their hydrodynamic diameter is increased compared to unmodified VLPs or capsids containing only CP.

Figure 2: Dynamic light scattering analysis. Hydrodynamic diameters of different P22-VLP species.

We also used ultracentrifugation over a sucrose cushion to separate freshly modified VLPs from monomeric capsid proteins, Sortase A5M, and sfGFP. After ultracentrifugation, a green fluorescent sediment was clearly visible (Fig. 3). This is a strong indication that sortase has attached sfGFP to the VLP exterior, as only assembled VLPs accumulate in the sediment. ^[8] We then prepared the ultracentrifugation sediment for transmission electron microscopy. Encouragingly, we observed numerous visually intact VLPs.

Figure 3: Sediment containing P22-VLPs modified with sfGFP using SortaseA5M. Sediment was imaged in transmission electron microscope.

References

1. ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [1]
2. ↑ W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nat. Struct. Biol. 2003, 10, 131. [2]
3. ↑ Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas, Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle ,ACS Chemical Biology 2014 9 (2), 359-365 [3]
4. ↑ Proft, T. (2010) Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation [4]
5. ↑ Mao, H., Hart, S. A., Schink, A., and Pollok, B. A. (2004) Sortase-mediated protein ligation: a new method for protein engineering [5]
6. ↑ Jean-Denis Pédelacq, Stéphanie Cabantous, Timothy Tran, Thomas C Terwilliger, Geoffrey S Waldo, Engineering and characterization of a superfolder green fluorescent protein, Nature Biotechnology volume24,pages79–88 (2006) [6]
7. ↑ FPbase: Superfolder GFP, last visited: 10.6.2019 [7]

Sequence and Features