Part:BBa_K3234000

Human interleukin 2

Interleukin-2 (IL-2) is a pleiotropic cytokine, and involves in immune regulation by controlling the differentiation and homeostasis of both pro- and anti-inflammatory T cells. As a naturally occurring cytokine made by T-lymphocytes, IL-2 stimulates the growth and activity of other T-lymphocytes and B-lymphocytes as part of the immune system. Many investigators explored IL-2 and/or IL-2-activated effectors for a variety of malignancies with encouraging but inconclusive results3. IL-2 has been approved by the U.S. Food and Drug Administration (FDA) to treat patients with stage IV (metastatic) melanoma since 1998. The sequence is codon optimized for expression in E coli.

Sequence and Features

Assembly Compatibility:

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  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
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  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 181
  

Usage and Biology

This is the coding sequence of human interleukin 2 (hIL2), which was constructed by us to test the expression capacity of hIL2 in E.coli. We tried to explore a suitable expression protocol to achieve efficient mass production of IL-2, and use these IL-2 to carry out our T cell proliferation and drug release tests.

The natural human IL2 sequence has a 60 bp signal peptide sequence at the 5' end, which is responsible for the secretion of hIL2 from human cells. In our design, this sequence is removed considering that the prokaryotic host can not recognize the signal peptide. Besides, Cys at No.125th of the original sequence may lead to the formation of an incorrect disulfide bond. So we mutate it to Ala to enhance the stability of the protein.

The lac operator at 5' end of the part allows the fermentation level to be controlled. When the fermentation progress is too fast, the protein may misfold into inclusion bodies, which will greatly reduce the yield of the protein. Therefore, quantitative regulation of fermentation is very important. What's more, the His tag was added to the 3' end to service for product purification with nickel column.

The goal of our project is to produce a fusion protein drug (hIL2+antiPDL1) that increases the immune level of cancer patients. We chose hIL2 as our protein drug, so it is necessary to measure and record the effect of interleukin 2 in treatment. With this part, we are able to produce pure interleukin 2 for further pharmacodynamic analysis experiments.

In order to explore an optimal expression host, optimal expression conditions (eg, inducer concentration, expression temperature, duration of induced expression, etc.). We designed multiple sets of different experiments to explore the effects of individual variables on experimental results.

Results

The SDS-PAGE results showed that a band corresponding to the size of the target band (about 16317 Da) appeared in the broken precipitate. This result indicates that our target protein forms inclusion bodies. We plan to express this part in yeasts and contine to try different expression conditions until we obtain the properly-folded protein.

MIT MAHE 2020

Summary

Interleukin-2 (IL-2) is an important immunological signal that influences various activities like differentiation, immuno responses and homeostasis. Stimulation with IL-2 is crucial for the maintenance of regulatory T cells and the differentiation of CD4+ T cells into defined effector T cell subsets following antigen-mediated activation. It is presented either in a soluble form or bound to dendritic cells and the extracellular matrix. It can with or without complexing with IL-2 specific antibodies amplify CD8+ T cell responses and helps in the upregulation of Treg cell population. Thus, they are involved in immune stimulation or suppression activities.

References

1. Boyman, O., Sprent, J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 12, 180–190 (2012). https://doi.org/10.1038/nri3157