High-level secretion of correctly processed beta-lactamase from Saccharomyces cerevisiae using a high-copy-number secretion vector.
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.
Castelli
L A
LA
Biomolecular Research Institute, Parkville, Victoria, Australia.
Mardon
C J
CJ
Strike
P M
PM
Azad
A A
AA
Macreadie
I G
IG
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004271:DNA, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D004271:DNA, Fungal; D001618:beta-Lactamases