June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Scalable production and testing of overexpressed TMEM97
Author Affiliations & Notes
  • Kyung-No Son
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Dhara Shah
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Sang Min Lee
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Marwan Ali
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Vinay Kumar Aakalu
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   Kyung-No Son None; Dhara Shah None; Sang Min Lee None; Marwan Ali None; Vinay Aakalu ViSo Therapeutics Inc., Code O (Owner), University of Illinois, Code P (Patent)
  • Footnotes
    Support  NEI/NIH R01EY024339, Department of Defense: W81XWH1710122, VA I01BX004080 Unrestricted Grant, Research to Prevent Blindness NEI P30EY001792
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3254 – A0289. doi:
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    • Get Citation

      Kyung-No Son, Dhara Shah, Sang Min Lee, Marwan Ali, Vinay Kumar Aakalu; Scalable production and testing of overexpressed TMEM97. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3254 – A0289.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : TMEM97 is an important endoplasmic reticular protein involved in cell migration, cancer, cholesterol processing, and neurodegenerative diseases. Histatins are a family of endogenous antimicrobial peptides that have numerous effects in multiple biological systems. Histatin-1 (Hst1) has roles in epithelial wound healing and migration. We recently showed, using biophysical and biological methods including co-immunoprecipitation, and surface plasmon resonance (SPR) that Hst1 is an endogenous ligand for TMEM97. We also determined that presence of TMEM97 was necessary for Hst1 to exert pro-migratory effects in corneal epithelia. Given the growing understanding that TMEM97 is important to ophthalmic biology, we sought to generate a method to scalably produce TMEM97 in an E. coli for future experiments. Commercially sourced TMEM97 is costly and is produced in eukaryotic systems. We validated the function of the E. coli expressed protein utilizing previously tested methods as well as a new orthogonal method [isothermal titration calorimetry (ITC)].

Methods : BL21DE3 cell culture pellets containing His-TEV-TMEM97 was processed and lysate underwent HisTrap affinity and SEC 16/600 SD200 purification. TMEM97 (108-176) was prepared in 10 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween-20. All ITC experiments were performed while stirring at 395 rpm using a VP-ITC titration microcalorimeter from MicroCal™, LLC (Northampton, MA). TMEM97 purchased from OriGene and His-TEV-TMEM97 in E.coli were prepared in HBS buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% n-dodecyl-β-D-maltoside (DDM). The CM5 sensor surface was first activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxy succinimide (NHS) mixture using a Biacore T200 or Biacore 8K instrument (GE Healthcare).

Results : We confirmed that Hst1 bound both commercially sourced and E. coli expressed TMEM97 utilizing. ITC was also performed as an orthogonal analytical method. Utilizing ITC we found that Hst1 bound TMEM97 with high affinity.

Conclusions : This study establishes a scalable research grade method for production of TMEM97, which demonstrates high-affinity binding to Hst1. Furthermore, we were able to demonstrate the binding between Hst1 and TMEM97 using a previously unreported method, ITC. This method will allow future research studies requiring large quantities of TMEM97 to better understand the importance of this protein to ophthalmic cellular function.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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