We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast. With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid. We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site. Large quantities of correctly-processed product can therefore be obtained. We show that 0.3 g/l of correctly processed beta-lactamase can be obtained in fed-batch cultures without the need for selective media or significant loss of the plasmid.
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