Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae

The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast c...

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Bibliographic Details
Published in:Biotechnology and bioengineering 1999-09, Vol.64 (5), p.607-615
Main Authors: Zhang, N, Gardner, D.C.J, Oliver, S.G, Stateva, L.I
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Biotechnology
cell lysis
Cell Wall - metabolism
Cells
Chemical operations
Chemical variables control
Chromosomes
cytolysis
DNA Primers
Fundamental and applied biological sciences. Psychology
Fungal Proteins - genetics
Genes
Genetic engineering
genetic regulation
Genetic technics
MET3 gene
Methods. Procedures. Technologies
Modification of gene expression level
PDE2
PDE2 gene
PKC1
PKC1 gene
Proteins
PSA1 gene
Recombinant Proteins - genetics
Saccharomyces cerevisiae
Saccharomyces cerevisiae - cytology
Saccharomyces cerevisiae - genetics
SRB1 gene
SRB1/PSA1
Yeast
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Summary:The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast cells in order to release their intracellular contents. To develop such a method, a tightly regulated promoter, pMET3, has been used to control three genes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of these regulation cassettes, pMET3-SRB1/PSA1 and pMET3-PKC1, have been integrated at the chromosomal loci of the respective genes in order to overcome problems of plasmid instability. Although repression of PDE2 did not cause cell lysis, cells depleted of Srb1p/Psa1p gradually lost their viability and integrity, releasing about 10% of total protein into the medium. Repression of PKC1 led to extensive cell lysis, accompanied by the release of 45% of cellular protein into the medium. A double mutant, carrying both pMET3-SRB1/PSA1 and pMET3-PKC1 cassettes in place of SRB1/PSA1 and PKC1, was constructed and found to permit the efficient release of both homologous and heterologous proteins.
ISSN:0006-3592
1097-0290
DOI:10.1002/(SICI)1097-0290(19990905)64:5<607::AID-BIT11>3.0.CO;2-0