In vitro tubule formation of Yop1 expressed in E. Coli

Abstract

The endoplasmic reticulum has three distinct regions: sheets of the nuclear envelope, peripheral ER sheets, and peripheral ER tubules. ER tubules are spread throughout the cell giving them contact with other organelles, which may allow for the transfer of lipids or for calcium signaling. The protein Yop1, found in S. cerevisiae, is one of several proteins necessary to maintain these ER tubules. For this project, Yop1 was conventionally cloned into pMCSG28 and then transformed into E. coli because of its ease of growth and genetic manipulation. The focus of this project is to determine an optimal expression and purification method of Yop1 from E. coli, while still yielding functional protein. Yop1 was purified with nickel affinity chromatography and size exclusion chromatography. Yop1 was tested for functionality by using transmission electron microscopy to see if it was able to form tubules in vitro.