Part:BBa_K3096044

Glucose dependent expression system of Exendin-4 with a N-terminal secretion tag and a CPP

This is the whole Expression System of Exendin-4. The system responds to glucose via the carbon catabolite repression system which initiates the transcription of the TetR gene when glucose levels are low. TetR binds to the TetR promoter inhibiting the Expression of Downstream genes, in this case our modified Exendin-4.

Background

In the iGEM Team Tübingen 2019 project, Exendin-4 was used as a therapeutic for the treatment of Type 2 Diabetes mellitus with probiotics. Exendin-4 is, in contrast to its human analogue more stable and not as easily degraded by DPP-IV peptidase. Thus, a suitable candidate for a therapeutic. This part is a composite, which allows for the glucose dependent secretion of Exendin-4 and a cell penetrating peptide -Penetratin- for the delivery of Exendin-4 into the circulatory system. If the glucose concentration is low, the bacterial Adenylate Cyclase is active and synthesises cAMP (cyclic AMP), which interacts with CAP (catabolite activating protein). The CAP-cAMP complex then binds the CAP binding site upstream of the TetR-gene, allowing for the expression of the Tet Repressor TetR, since the transcription of the repressor is activated. The TetR accordingly binds and blocks the activity of the Tet repressible promoter, which controls the expression of our GOI construct. If the glucose availability increases after the intake of a meal, Adenylate Cyclase is inhibited and the CAP-cAMP complex dissolves. Therefore, the TetR is not active and the GOI can be expressed.

Usage and Biology

Exendin-4 is used as a treatment for Type 2 Diabetes mellitus. Its human analogue gLP-1 originates from the prepeptide proglucagon, which is cleaved into, among others, GLP-1 (1-37) by prohormone convertase 1 (PC1) [Lim et al. 2006]. The inactive GLP-1 (1-37) is then processed into its active form GLP-1 (7-37) by an endopeptidase. This active form is secreted and diffuses across the basal lamina and enters the lamina propria, where it is taken up into the capillaries, reaching the circulatory system [Lim et al. 2006].The glucagon like peptide binds to a GPCR on the pancreatic beta cells, which will cause an intracellular cascade activating an Adenylate Cyclase. If ATP is available within the cell, which means if the cell has already taken up sugar and run through glycolysis, cAMP will be produced [Jens Juul Holst, 2007]. Overall, this will result in prolonged depolarization and calcium ion influx and consequently more secretion of insulin from the beta cells [Jens Juul Holst, 2007]. This increase in insulin secretion can overcome the relative lack of insulin in the periphery of Type 2 Diabetes mellitus[Jens Juul Holst, 2007].

Exendin-4 and GLP-1 do not only have a direct effect on the pancreas, but also decrease gastrointestinal motility and secretion, hunger and the emptying of the stomach, thus helping in weight reduction [Jens Juul Holst, 2007]. Additionally, they are considered to be cardioprotective [Jens Juul Holst, 2007]. This is very important for the therapy, since many cases of Type 2 DM are caused by an unhealthy lifestyle and obesity [Jens Juul Holst, 2007].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1297
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Holst, Jens Juul. The physiology of glucagon-like peptide 1. Physiological reviews 87.4 (2007): 1409-1439.
2. Lim, Gareth E., Brubaker, Patricia L. Glucagon-Like Peptide 1 Secretion by the L-Cell. (2006). 10.2337/db06-S020. Diabetes. p. S70-S77
3. Copley, Kathrin, et al. "Investigation of exenatide elimination and its in vivo and in vitro degradation." Current drug metabolism 7.4 (2006): 367-374.