Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.
Bottom Line: The production of milligram quantities of recombinant protein is still a serious obstacle to the structural and functional characterization of these proteins.We further demonstrate that the increase in expression for our test proteins resulted in a concomitant increase in functional protein.Using this system, we were able to increase the expression level of a plant transporter, NRT1.1, which was a key factor in its structural and functional characterization.
Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.Show MeSH
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Mentions: To improve the yield of recombinant integral MPs produced in S. cerevsiae, we introduced a LEU2 gene containing a truncated version of its own promoter into the pDDGFP2 vector12 at the unique NaeI site [Fig. 1(A)]. The pDDGFP2 vector was chosen as it was used previously to over express a number of eukaryotic MPs.12 The pDDGFP2 vector contains the strong and inducible GAL1 promoter, which is used to drive expression of the gene of interest, following addition of galactose as the carbon source during culturing. The recombinant MP is produced as a C-terminal yeast enhanced GFP fusion containing an octa-histadine affinity purification tag, this tag can be removed due to the presence of a Tobacco Etch Virus (TEV) protease site upstream of the GFP.19 The pDDGFP2 vector also allows for ligation free cloning through the use of regions flanking a unique SmaI site, which can be used to insert PCR products via homologous recombination through cotransformation of both PCR and SmaI lineralized vector into chemically competent yeast cells.
Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.