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.