A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity.
A system has been developed for the release of heterologous proteins from Saccharomyces cerevisiae, based on the use of thermosensitive osmotic-remedial mutants, deficient in cell integrity, that lyse at the non-permissive temperature, thus releasing the bulk of intracellular proteins and leaving behind cell ghosts and debris. The strains developed combine the lyt2 mutation (which is allelic to gene SLT2/MPK1 coding for a MAP kinase homolog), with the disruption of genes PEP4 and PRB1 known to produce a protease-deficient background. Cells transformed with the appropriate bacterial gene, released about 70% of the heterologous protein chloramphenicol acetyl transferase (CAT) in bioreactor cultivation upon switching growth temperature to 37 degrees C, or by osmotic shock of the cells preincubated at 37 degrees C in the presence of 1 M sorbitol. It is suggested that our release system could be advantageous for obtaining large-scale protein preparations for downstream processing without any mechanical breakage of the cells, enzymatic treatment or chemical extraction.
Alvarez
P
P
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Sampedro
M
M
Molina
M
M
Nombela
C
C
D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015500:Chloramphenicol O-Acetyltransferase; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae