Team:BNDS China/Design

Design


Calcium release-activated calcium modulator 1 (Orai1) is our part (BBa_K2028000) this year.

Orai1 is one of the key components in the Calcium release-activated channels (CRAC)channel signaling machinery. The channel also composed of a protein called stromal interaction molecule 1 (STIM1) acts as an endoplasmic reticulum-located Ca2+sensor and mediates CRAC channel activation. After endoplasmic reticulum Ca2+store depletion, STIM1 multimerizes and redistributes into punctae close to the plasma membrane. It undergoes a switch to an extended conformation, thereby coupling to the channel protein Orai. Finally, the coupling of STIM1 to Orai culminates in CRAC channel activation, which allows Ca2+ influx into the cell. However, research done by Beth A. McNally et al. suggested that the mutation of Orai1 at the residue 102 from Valine into Alanine produced constitutively active CRAC channels that were open even in the absence of STIM1. In order to reduce the complexity and fussiness of our project, we chose the mutant of Orai1V102A as our part. We cloned Orai1 V102A into the expression vector pGEX-KG and are going to express this protein in bacteria BL21(DE3).
By using Orai1 V102A as our part, the procedures and time have reduced.

Reference

  • McNally, Beth A. et al. "Gated Regulation Of CRAC Channel Ion Selectivity By STIM1". Nature 482 (2012): 241-245. Web. 19 Oct. 2016.
  • Rosado, Juan A. et al. "STIM And Orai1 Variants In Store-Operated Calcium Entry". Frontiers in Pharmacology 6 (2016): n. pag. Web. 19 Oct. 2016.

  • Derler, I. et al. "The Extended Transmembrane Orai1 N-Terminal (ETON) Region Combines Binding Interface And Gate For Orai1 Activation By STIM1". Journal of Biological Chemistry 288.40 (2013): 29025-29034. Web. 19 Oct. 2016.