Coding

Part:BBa_K2616000

Designed by: iGEM Pasteur Paris 2018   Group: iGEM18_Pasteur_Paris   (2018-09-05)


proNGF and TEV secretion via E. coli Type I Secretion System

This part permits to secrete proNGF directly in the extracellular medium using E. coli type I secretion system. We used inducible promoter T7, in order to control proNGF production thanks to IPTG induction. We also added an His-tag in N-terminal in order to purify it. proNGF is adressed to Type I Secretion System by fusing to it the the 60 C-terminal aminoacid of alpha-haemolysin HlyA. Since the export peptide is not processed when passing through the secretion pore, we separated proNGF from this 60 aminoacid long sequence by the cleaving site for TEV protease ENLYFQ. This part also permits to secrete TEV protease, under the same promoter, via this same secretion system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 62
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 430
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1845
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1831

Cloning



This DNA construct was ordered in two parts, named Seq1 (1096 bp) and Seq2 (1153 bp) in commercial plasmids pEX-A258 from gene synthesis. Seq1 and Seq2 were amplified in competent E. coli DH5-α. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (NheI and BamHI for Seq1, MscI and HindIII for Seq2). We extracted the inserts from the gel and performed a ligation by using specific overlaps into linearized pET43.1a for proNGF expression and into pSB1C3 for iGEM sample submission. We proved that our vector pet43.1a contained Seq1 and Seq2 (Figure 1) and that pSB1C3 contained Seq1 and Seq2 (Figure 2) after digestion and DNA electrophoresis. Plasmid DNA of pSB1C3 construction was purified and sent for sequencing.


Figure 1: Agarose 1% gel after electrophoresis of digested pET43.1 containing Seq1 and Seq2 (Bba_K2616000) with NdeI. Colonies 6, 9, 10 ,11, 12 and 15 have the correct construction.

Figure 2: Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq1 and Seq2 (Bba_K2616000) with EcoRI/PstI. Colonies 3, 7 and 8 have the correct construction.


Alignment of sequencing results confirmed that pSB1C3 contained Bba_K2616000


Figure 3: Alignment of sequencing results for BBa_K2616000. Sequencing perform in pSB1C3 and three primers were designed (FOR1, FOR2, FOR3) to cover the whole sequence. Image from Geneious.

The construction was successfully assembled. In Figure 3, we show that we used three different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing. The final basepair identity is 100%

proNGF characterization and purification



Our chassis is Escherichia coli BL21(DE3) pLysS, a specific strain dedicated to producing high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with BBa_K2616000 and pVDL 9.3, generously provided by Dr. Victor de Lorenzo, from Centro Nacional de Biotecnología of Madrid, bearing HlyB and HlyD (Type I secretion system) sequences, in order to get a chance to secrete proNGF out of the cell.

Bacteria were grown at a large scale (800 mL), and proNGF expression was induced with 0.1 mM IPTG for 2 hours at 37°C.

We tried to achieve His-tagged proNGF purification using a single step Ni-NTA affinity purification column. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected (fraction A6).


Figure 4: FPLC proNGF purification with ÄKTA pure (General Electric) Ni-NTA column was equilibrated with buffer A (50 mM Tris, pH 7.4, 200 mM NaCl). Supernatant of lyzed bacteria was introduced through the column. Washing with 5% of buffer B. Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, and concentration of buffer B in green.

We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry.


Figure 5: SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.



The proNGF purification using a single step Ni-NTA column was not conclusive. Many proteins were found on elution fractions. His-tagged proNGF fused to HlyA export signal should be found at 33 kDa while the proNGF cleaved by TEV protease should be found at 27 kDa. We finally analyzed five gel slices around 20 to 35 kDa of the FPLC flow-through (lane 2, Figure 5) by LC/MS/MS mass spectrometry, to verify the presence of proNGF.

With the LC/MS/MS analysis, 14 coverage unique peptides corresponding to proNGF were found in all fractions. The sequence coverage represents 63%. Results of mass spectrometry analysis demonstrate the expression of proNGF According to Figure 6, proNGF pattern are found on each fraction sent to mass spectrometry. The major amount is found on fraction 5, corresponding to 33 kDa. At this molecular weight, the proNGF is still fused to the signal peptide. However these results are also consistent with a mix of cleaved and uncleaved proNGF. The TEV protease, 34 kDa fused to export singal and 28 kDa cleaved from the export signal are found.


Figure 6: Distribution of matching peptides of proNGF and TEV protease by gel fractions after mass spectrometry analysis.

Analysis of Fraction 5 of the gel shows that our protein proNGF is present in a mix of cleaved and uncleaved polypeptide (Figure 7). Mass spectrometry spectrum of Peptide A, IDTACVCVLSR, from proNGF sequence and peptide B IISAAGSFDVKEER from fused HlyA export signal are shown in Figure 8. The presence of mass spectrometry identified peptides corresponding to the fusion of proNGF and HlyA indicate some proNGF uncleaved from the export signal.

Figure 7: Alignment sequences of proNGF fused to HlyA export signal and peptides identified by mass spectrometry. In light blue peptides that match proNGF amino acids sequence. In light yellow, peptides that match HlyA export signal. Sequence has been annotated to match corresponding protein amino acid sequences : In orange His tagged proNGF, in red TEV protease cleaving site, in pink HlyA export signal.

Figure 8: Mass spectrometry spectrum. A) Peptide identified corresponding to proNGF. B) Peptide identified corresponding to the fusion of proNGF and HlyA export signal.


The proNGF did not seem to be retained on the Ni-NTA affinity column, although in fraction A6 we also identified His-tag bound proNGF. To test if the His-tag is accessible for binding to Ni-NTA, we've performed a batch purification using Ni-NTA beads under native and partial denaturing conditions (Urea 2 M) followed by Western Blot analysis with immunodetection through Anti-His Antibodies Alexa Fluor 647 (Figure 9). Detection of His-tag in the pellet supernatant of induced BL21(DE3) pLysS with 1 mM IPTG and flow through when partially denatured.

Native His-tagged proNGF was not retained on Ni-NTA beads. We believe that the N-terminal His-tag may be hidden in the protein fold. Consequently, we denatured with 2M urea before purifying on the beads. As seen in lane 8 even 2M urea could not improve the binding. We also tried with an 8M urea concentration, without better results.

Figure 9 : Western Blot analysis of batch purification of proNGF under native and partial denaturing conditions.


Usage and Biology

Effect of produced proNGF on In vitro neural primary culture from E18 Sprague Dawley cortexes :



After having collected the data on the effect of commercial NGF(see [http://2018.igem.org/Team:Pasteur_Paris/Results]), we decided to put in culture our cells in the presence of our bacterial lysate to test the effect of our proNGF. We put in culture for 2 days 30 000 cells with or without commercial NGF at 500 ng/mL and 900 ng/mL as well as our bacterial lysate in different dilutions. Since we wanted to inactivate as much bacterial proteins as possible (endotoxins), we checked the denaturation temperature for our proNGF, 70°C, and heat-inactivated the lysate at 60°C for 5 minutes before putting it in culture. Due to lack of time, only one well per condition was analyzed.


Figure 10: (A) Percentage area of β-III Tubulin in each well and (B) percentage area of nucleus in each well with no commercial NGF, 500 ng/mL or 900 ng/mL or bacterial lysate at 1/5, 1/10, 1/20 or 1/30 added in our medium Neurobasal, B27, GlutaMAX.

We can see in Figure 10 that our lysate seems to increase the percentage area of the β-III Tubulin compared to the control without NGF. Our results with the commercial NGF seem to be equivalent to the results we had from our first experiment, with a decrease of axons at a concentration of 900 ng/mL. We can hypothesize that the lysate does have an effect on axon’s growth from the increasing percentage area of β-III Tubulin, increase similar to the one we observe in our previous experiment with commercial NGF. Thus, the activity of our proNGF could be equivalent to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.

We also could see an influence of the commercial NGF on the survival of the cells, similar to our previous experiment with commercial NGF . Our lysate, put at a concentration of 1/10 and higher, seems to have the same effect (Figure 11).


Figure 11: Image of the whole well of the 96-well plate. Neurons were put in culture in Neurobasal, B27, GlutaMAX, and our lysate at a concentration of 1/10 medium

Those data require further statistical tests, since we only had time to analyze one well per condition, and for only 2 days of culture due to French customs administrative delays that came with the order of the E18 cortex pair from the USA. Still, in those 2 days of culture, we have been able to observe a difference in both the percentage area of β-III Tubulin and nuclei counts.

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