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

Profile

Name	TVMV-GGGG-mCherry
Base pairs	1330
Molecular weight	29.4 kDa
Origin	synthetic, derived from Discosoma sp.
Parts	mCherry, GGGG-Sequence, TVMV site, T7 Promoter, lac Operator, RBS, araBAD promoter + RBS, GASPAG Linker, Strep-Tag II, Double Terminator for pDEStara2
Properties	Red fluorescent, Ex λ: 587nm, Em λ: 610 nm

Usage and Biology

mCherry (BBa_K3187026) is a red fluorescent protein. Which is a synthetic protein derived from Discosoma sp. by directed evolution. The N-terminal GGGG-sequence (BBa_K3187018) can be fused to a protein with a C-terminal LPETGG-Sortase A link (BBa_K3187019) by Sortase A. In order to remove the first methionine in front of the GGGG-Sequence a TVMV restriction site is cloned in the sequence. By removing the first methionine the linkage of LPETGG and GGGG-Sequences should work better. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pDEStara2 vector, containing the sequence of mCherry, a GGGG-sequence, a TVMV restriction site (BBa_K3187020), a GASPAG-Linker (BBa_K3187038), a Strep-Tag II (BBa_K3187025) for Purification, a T7 promoter with lac-operator and an RBS (BBa_K3187029), a T7TE terminator (BBa_K3187032), a Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029). Since pDEStara2 is a vector for dual expression it also contains an araBAD promoter with an RBS (BBa_K3187041) and a Double Terminator (BBa_K3187042). The coding sequence consists of 851 bp which are translated to 260 amino acids.^[1]

Methods

Purification

The GGGG-mCherry with TVMV Restrictionsite 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-Tag II. TVMV protease cleavage was performed over night at 4 °C.

Results

Does methionine affect Sortase linking?

Sortase A7M preferably attaches N-terminal poly-G to C-terminal LPETGG. However, the first amino acid of a protein is methionine (to be specific, formylmethionine in bacteria). For our constructs that possess N-terminal polyG-tags, we have to ask ourselves the question: If the initial methionines are not cleaved off after the proteins have been produced, will this interfere with the Sortase reaction?

To investigate this, we cloned and purified another protein: TVMVsite-GGGG-mCherry. This protein can be treated with TVMV-protease, leading to *GGGG-mCherry. This *GGGG-mCherry was then compared to (M)GGGG-mCherry we used in all previous assays.

Also, we tested if it is possible to also link another substrate with another protease cleavage site infront of the N-terminal GGGG-sequence also aplicable. Fig. XY shows that this is possible. Due to these findings we modified our VLPs with *GGGG-sfGFP.

Figure x : Sortase-mediated ligation of TAMRA-LPETG and GGGG-sfGFP (with TEV cleavage site) one cut with TEV protease and one not. The sample with the unprocessed substrate shows no increase in RFU. In contrast the processed substrate shows a clear increase in delta RFU. After 90 min the reverse reaction begins.

We performed FRET-assays with TAMRA-LPETG and either of the following reaction partners:

(M)GGGG-mCherry, a protein sample that might still carry an N-terminal methionine
*GGGG-mCherry that does not carry any additional N-terminal residue

Before the FRET-assay was started, we adjusted the mCherry-concentrations of both fluorescent protein solutions to the same level. To do so, we diluted them until both showed the same fluorescence at 610 nm.

Figure x: FRET of the sortase reaction connecting TAMRA-LPETG and GGGG-sfGFP mediated by Sortase A7M. The concentration of the Sortase A7M was kept at the same level why the concentration of sfGFP was either 7.8 mM or 1 mM. The graphs show that the reverse reaction happens earlier if if the GGGG-substrate concentration is lower.

Strikingly, only the (M)GGGG-mCherry construct showed a clear decrease in delta RFU after the maximum delta RFU was reached (at about XXX min).

Figure x : Sortase-mediated ligation of TAMRA-LPETG and GGGG-mCherry one cut with TVMV protease and one with a methionin infront of the GGGG-tag. As visible the reverse reaction happens earlier if the methionine is not cleaved of the GGGG-tag. The delta RFU is referring to the negative controls without Sortase A7M.

We assume the following: Although we adjusted the overall mCherry concentration by fluorescence, we cannot determine the absolute amount of MGGGG-mCherry in the (M)GGGG-mCherry sample. However, if this amount was relatively high, the effective substrate concentration that could enter the sortase reaction would be low. That is because MGGGG is a worse sortase substrate than GGGG – if any at all. If we furthermore consider that a low substrate concentration correlates with a faster reverse reaction, we can explain the observed decrease in delta RFU for the (M)GGGG-mCherry sample that contrasts the delta RFU trend of the *GGGG-mCherry sample.

On this basis we can assume that a certain, yet unknown portion of the (M)GGGG-mCherry sample still carries an N-terminal methionine.

These FRET-assays let us assume that methionine disturbs or at least interferes with the sortase reaction mechanism. Indeed, our modeling suggests that methionine affects the interaction of polyG and the flexible loop near the active site of Sortase A7M. Click here if you want to know more about our modeling results!

This strengthens our hypothesis: If there is any amino acid in front of the poly-glycine sequence, substrate binding to Sortase A7M is negatively influenced.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1197
Illegal PstI site found at 1133
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 239
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 324
Illegal PstI site found at 1133
Illegal AgeI site found at 74
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 56