The yeast Saccharomyces cerevisiae has been used as a production organism for recombinant proteins for many years. One of the most important characteristics of organisms used for protein production is their secretion capacity, as secretion facilitates the recovery of produced proteins. In this master’s thesis, we aimed to develop a high-throughput, semi-quantitative method for assessing the protein secretion capacity of S. cerevisiae cells. First, a suitable enzyme had to be selected as a model that would enable protein secretion to be monitored on solid agar media and secretion capacity to be semi-quantitatively evaluated based on the enzyme activity. An amylase proved to be the most suitable enzyme, as its activity can be monitored on starch-containing media by the formation of a clearance zone following exposure to iodine vapour. Subsequently, a gene cassette, containing the selected amylase was assembled by PCR and integrated into the yeast genome at the LYP1 locus by homologous recombination. This was followed by mating of a strain which contained the assembled gene cassette, with a strain, which did not contain the gene cassette. The resulting segregants were screened for amylase activity and, in the final stage of the experiment, compared to identify the segregant with the highest capacity for heterologous protein secretion. The developed method showed promising results; however, further research and experiments are required to optimize the method and confirm the applicability of the obtained results.
|
