13: Protein overexpression

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In this lab, you will use various carbon sources to manipulate the expression of Met and LacZ fusion proteins in
cells that have been transformed by overexpression plasmids. The Gal4p transcription factor (above) binds to
the GAL1 promoter in the plasmids and controls protein expression. In this lab, you will prepare protein
extracts from cells growing under both repressed and induced conditions for later analysis.

Objectives

At the end of this lab, students will be able to

Compare the effects of different carbon sources on transcription of genes controlled by the yeast GAL1 promoter.
Explain the roles of heat and detergents in the preparation of yeast cell extracts.
Culture yeast with different carbon sources to induce or repress expression from the GAL1 promoter.
Prepare extracts from transformed yeast strains suitable for use in SDS-PAGE gels and western blots.

Over the next few weeks, you will be analyzing the expression of S. pombe and S. cerevisiaeMet or bacterial LacZ fusion proteins in your transformed strains. You have already tested the ability of the overexpression plasmids to complement the met mutation in your yeast strains. Complementation depends on the presence of functional Met proteins. If you observed a failure to complement the met deficiency, this could indicate that proteins were not expressed from the plasmids. Alternatively, the overexpressed proteins may be present, but not function normally.Remember that the proteins expressed from the BG1805 and pYES2.1 plasmids are fusion proteins with additional sequences at their C-termini (Gelperin et al., 2005). The biochemical activities of these fusion proteins have not been previously evaluated. (We are the first to test whether the Met fusion proteins function similarly to the normal S. cerevisiae proteins!)

In this experiment, you will prepare extracts from yeast for later experiments (Chapters 14 and 15) in which you will determine if the Met and LacZ fusion proteins are being successfully expressed in the transformed yeast strains and how the expression of the fusion proteins varies with carbon sources.