Expression and purification of the glycosylated Flo1 protein of Saccharomyces cerevisiae.

Yeast cells possess a remarkable capacity to adhere to other cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell-interactions are mediated by a class of special cell wall proteins, called flocculins that stick out of the cell walls of flocculent cells and selectively bind mannose residues present in the cell walls of adjacent yeast cells. For brewers, flocculation is a desirable property of industrial Saccharomyces cerevisiae strains as it allows the easy separation of cells from the fermentation product. Only at the end of the fermentation process, the yeast cells start adhering to each other to form macroscopic 'flocs' consisting of many thousands of cells and will sediment. Flocculins consist of three domains. The first one is the mannose binding domain. The middle domain consists of a variable number of repeats and is heavily glycosylated. The protein is anchored to the cell wall by the C-terminal domain.
In this work, we studied the Flo1-protein, which is the most important flocculin responsible for flocculation of yeast cells. The final goal is the characterization of the binding mechanism of the protein to mannose. As the mannose binding site is situated in the N-terminal domain, this domain is the most interesting one to study. Therefore, it was cloned with a C-terminal histidine tag in Saccharomyces cerevisiae using the pYEX-S1-vector. As this vector contains a secretion sequence, the proteins were purified from the medium using a nikkel column followed by gel filtration. It was shown that the proteins are heavily glycosylated and that the proteins are aggregating due to the glycans. The binding assay showed that deglycosylated proteins were able to bind stronger to mannose beads than glycosylated proteins.