Latest revision as of 18:26, 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
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. The vector posesses a kanamycin resistance. Expression is controlled by the T7 promoter and induced by IPTG.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 produce the N-terminal polyG tag. The cleaved protein was then used in different characterisation assays of different Sortase variants.

The measured absorbance and emission spectra indicated that TAMRA and superfolder green fluorescence protein (sfGFP) are a possible FRET-pair. The sfGFP has an N-terminal polyglycine sequence and can therefore be linked to TAMRA with the sorting motif, in the same way as mCherry was connected. However, the small overlap between the extinction spectra of sfGFP and TAMRA could solve the previous “simultaneous excitation” problem we observed for the mCherry-TAMRA FRET-pair. Because of the lower excitation maximum of sfGFP compared to TAMRA, sfGFP was chosen as donor and TAMRA as acceptor. sfGFP was excited at 465 nm to minimize the unnecessary leak excitation of sfGFP.

Figure 2 : Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-sfGFP (sfGFP). In this configuration sfGFP acts as donor and TAMRA as acceptor. When the two fluorophores are not linked only sfGFP is being excited. After sortase-mediated ligation of the two substrates, TAMRA is the fluorophore being excited via FRET and the emission of TAMRA intensifies. Meanwhile, the emission of sfGFP decreases.

The transfer of energy from sfGFP to TAMRA can be seen by the decrease in emission of sfGFP and increase in emission from TAMRA. Compared to TAMRA as an acceptor, the sfGFP bleaches significantly less and is consequently more suitable as a donor for FRET. Furthermore, the afore mentioned problem of simultaneous donor and acceptor excitation seems to be solved. It seems that we have found a FRET-pair with superior properties.

Figure 3 : Spectrum of TAMRA and sfGFP, with Sortase A7M, over the course of 25 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.

Due to the collected data of both FRET-pairs we decided to use the TAMRA-LPETG and GGGG-sfGFP FRET-pair for further characterization of our Sortase A variants. Two reasons justify this decision:

TAMRA bleaches stronger than sfGFP when excited with a laser.
The spectral overlap between TAMRA and mCherry disturbs “clean” energy transfer, thus the FRET-effect would be less visible and could not be used for analysis of the sortase-mediated reaction.

For recording of sortase reaction parameters we recommend using the FRET-pair sfGFP-TAMRA. As this pair of fluorophores proved to have near perfectly aligned spectra and since the bleaching effect is visibly lower on sfGFP than on TAMRA, we chose to use this FRET-pair in most of our following assay. Nevertheless, we do not rule out the use of TAMRA-mCherry as a FRET-pair since we used it in several FRET-assays as well.

Figure 4 : Animation of Sortase A7M enzyme kinetics over the course of 3 h. The reaction speed increases radically in the beginning moving from RFU 8000 to RFU 6000 at λ = 550 nm where a plateau is reached (blue). The negative control (orange) is also reduced in its RFU due to bleaching. Nevertheless, a peak at λ = 580 nm arises already after short reaction time. This peak indicates the successful Fluorescence Resonance Energy Transfer.

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.

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