Coding

Part:BBa_K4764001

Designed by: Katie Spivakovsky   Group: iGEM23_MIT   (2023-10-08)


3xFLAG-IL6-mCherry fusion protein

This linear sequence expresses a fusion protein containing interleukin-6 tagged on its 5' end with a 3xFLAG tag for purification and on its 3' end with mCherry for red fluorescence. It can be used to transfect cells to produce IL-6 that can then be purified and identified via its red fluorescence, making it useful for a variety of experiments.

A central component of our experiment rested on the assumption that we would be able to purify our 3xFLAG-IL6-mCherry fusion protein from HEK293 cell lysate with extremely high yield. First and foremost, we needed to optimize the transfection efficiency of our fusion protein. The below photo shows the transfection of HEK293 cells at passage 20 with 600 ng of plasmid DNA at a 48 hour timepoint. p20mcherry.png

At this point, we began to approach protein purification from a few different perspectives.

Initially we considered biotinylating our protein to fluorescent streptavidin silica beads. This would knock out three birds with one stone:

  1. We could create IL-6 clusters of variable, controlled sizes on a micron scale to surpass the size and signal threshold of macrophages
  2. We could isolate the IL-6 protein from the rest of the HEK293 cell lysate
  3. We could detect via fluorescence if the macrophages only bound to the IL-6 or actually phagocytosed the whole bead

However, we decided against the bead idea because it would require multiple rounds of purification, with unknown and likely decreasing protein yield rates. We valued the quantity of final protein above the existence of IL-6 aggregates for our proof-of-concept

We instead turned to simply running one round of anti-FLAG tag purification. We utilized Pierce anti-FLAG magnetic agarose beads to selectively isolate the 3xFLAG-tagged IL6. We optimized the protocol by varying over three elution buffers:

  • IgG Elution Buffer
  • 0.1 M glycine
  • SDS-PAGE sample loading buffer, namely 1x Laemmli buffer

    • In order to give us an idea of our protein yield rate, we turned to using SDS-PAGE gel electrophoresis, the Bradford assay, and the NanoDrop spectrophotometer. At first, we had hoped that the SDS-PAGE gels would be able to verify the existence of our protein (by length) and the relative concentration (by band color intensity). After dozens of attempts to optimize the SDS-PAGE equipment available to us, we concluded that it would be an unreliable form of measurement. Then, we looked to quantify our protein concentration via Bradford assay and absorbance comparisons to bovine serum albumin standards. We were getting readings extremely close to 0 Abs and even negative absorbances, and we verified this tragic data with the NanoDrop.

    Regardless of this insanity check, we proceeded with attempting to incubate the so-called “purified protein elution” with our CAR-expressing HEK293 cells. After concluding that there was no red fluorescence detected after incubation, we reconsidered our purification procedure. Noor, one of our mentors, brought up the idea of incubating the CAR-expressing HEK293 cells directly with the lysate of IL6-expressing HEK293 cells. We examined the lysis and purification steps that we had been applying—so far, we had been lysing the IL6-expressing HEK293 cells with ThermoFisher M-PER reagent. Since M-PER was a detergent that would unintentionally lyse our CAR-expressing cells and could not be extracted from the final lysate solution, we turned to another form of mammalian cell lysis to accomplish our goal: freeze-thaw lysis. The final lysate was not purified; we directly added it to wells of CAR-expressing HEK293 cells to observe the resulting behavior. This way, we knew IL-6 was in the lysate and would no longer act as a limiting factor.

    We followed the 2013 Calgary iGEM team’s protocol (https://2013.igem.org/Team:Calgary/Notebook/Protocols/FreezeThawProtocolforProteinSamples) but for a 10 cm dish of HEK293 cells. Ultimately, after viewing the fluorescence under our Keyence BZ-X810 microscope using the GFP and RFP filter cubes, we saw significantly more red fluorescence with freeze-thaw lysate than the purified protein eluted with Laemmli buffer and the purified protein eluted with IgG buffer.

    A photo of co-culturing with freeze-thaw lysate is below. coculture1.png

    A photo of incubating with IgG-eluted IL-6 is below. cocultureigg.png

    Sequence and Features


    Assembly Compatibility:
    • 10
      INCOMPATIBLE WITH RFC[10]
      Illegal XbaI site found at 549
      Illegal PstI site found at 529
      Illegal PstI site found at 604
      Illegal PstI site found at 625
      Illegal PstI site found at 673
      Illegal PstI site found at 1078
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal PstI site found at 529
      Illegal PstI site found at 604
      Illegal PstI site found at 625
      Illegal PstI site found at 673
      Illegal PstI site found at 1078
    • 21
      COMPATIBLE WITH RFC[21]
    • 23
      INCOMPATIBLE WITH RFC[23]
      Illegal XbaI site found at 549
      Illegal PstI site found at 529
      Illegal PstI site found at 604
      Illegal PstI site found at 625
      Illegal PstI site found at 673
      Illegal PstI site found at 1078
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal XbaI site found at 549
      Illegal PstI site found at 529
      Illegal PstI site found at 604
      Illegal PstI site found at 625
      Illegal PstI site found at 673
      Illegal PstI site found at 1078
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal SapI site found at 691


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