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Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

Parker JL, Newstead S - Protein Sci. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

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A modified S. cerevisiae expression system for eukaryotic MP production. (A) Vector map of pDDGFP2-Leu2d vector. The vector allows for dual selection using either the uracil or leucine markers. The gene for the recombinant protein is inserted via homologous recombination into the smaI site and is, then, expressed under the control of the GAL1 promoter as a tev cleavable C terminal tagged GFP-His fusion. (B) Comparison of expression level between the use of either the URA3 (selection using medium lacking uracil) or LEU2 genes (selection using medium lacking leucine) as selective markers for a number of different membrane proteins (see Table I for details). (C) As (B) but looking at yeast strain dependence of the system for the expression of two constructs.
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fig01: A modified S. cerevisiae expression system for eukaryotic MP production. (A) Vector map of pDDGFP2-Leu2d vector. The vector allows for dual selection using either the uracil or leucine markers. The gene for the recombinant protein is inserted via homologous recombination into the smaI site and is, then, expressed under the control of the GAL1 promoter as a tev cleavable C terminal tagged GFP-His fusion. (B) Comparison of expression level between the use of either the URA3 (selection using medium lacking uracil) or LEU2 genes (selection using medium lacking leucine) as selective markers for a number of different membrane proteins (see Table I for details). (C) As (B) but looking at yeast strain dependence of the system for the expression of two constructs.

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.


Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

Parker JL, Newstead S - Protein Sci. (2014)

A modified S. cerevisiae expression system for eukaryotic MP production. (A) Vector map of pDDGFP2-Leu2d vector. The vector allows for dual selection using either the uracil or leucine markers. The gene for the recombinant protein is inserted via homologous recombination into the smaI site and is, then, expressed under the control of the GAL1 promoter as a tev cleavable C terminal tagged GFP-His fusion. (B) Comparison of expression level between the use of either the URA3 (selection using medium lacking uracil) or LEU2 genes (selection using medium lacking leucine) as selective markers for a number of different membrane proteins (see Table I for details). (C) As (B) but looking at yeast strain dependence of the system for the expression of two constructs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230410&req=5

fig01: A modified S. cerevisiae expression system for eukaryotic MP production. (A) Vector map of pDDGFP2-Leu2d vector. The vector allows for dual selection using either the uracil or leucine markers. The gene for the recombinant protein is inserted via homologous recombination into the smaI site and is, then, expressed under the control of the GAL1 promoter as a tev cleavable C terminal tagged GFP-His fusion. (B) Comparison of expression level between the use of either the URA3 (selection using medium lacking uracil) or LEU2 genes (selection using medium lacking leucine) as selective markers for a number of different membrane proteins (see Table I for details). (C) As (B) but looking at yeast strain dependence of the system for the expression of two constructs.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

Show MeSH
Related in: MedlinePlus