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.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Alvarez P, Sampedro M, Molina M, Nombela C

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