Overexpression of SERCA1a Ca2+-ATPase in yeast.

Abstract

Much effort has been invested to express the SERCA1a Ca2+-ATPase, a large membrane protein that transports Ca2+ across the sarcoplasmic reticulum membrane. Various expression systems have been set up with that goal, including COS cells1,2 and Spodopter fugiperda Sf93 or Sf214 cells, via baculovirus and yeast,5–7 all giving a relatively low amount of material. Here, we have optimized a yeast expression system8 for the production in yeast of high amounts of a C-terminal His-tagged Ca2+-ATPase, using a galactose-regulated promoter. The expression was improved by optimizing the number of galactose inductions and modifying the host yeast to increase the amount of the Gal4p transcription factor, which controls the promoter behind which the SERCA gene was inserted. It was necessary to reduce the temperature of expression from 28°C to 18°C in order to enhance recovery of solubilized Ca2+-ATPase, which could then be fully solubilized with L-α-lysophosphatidylcholine or by 50% with n-dodecyl-β-D-maltoside. FIGURE 1 displays an electron-microscope picture that shows that the expressed protein is located mainly in periendoplasmic membranes. A 4-L culture produced 100 mg of Ca2+-ATPase, with 60 and 22 mg being pelleted with the heavy and light membrane fractions, respectively. The expressed Ca2+-ATPase was further purified by metal affinity chromatography as described by Lenoir et al. elsewhere in this volume.9