WO1996002629B1

WO1996002629B1 - Process for releasing heterolog proteins of saccharomyces cerevisiae strains

Info

Publication number: WO1996002629B1
Application number: PCT/ES1995/000088
Authority: WO
Filing date: 1995-07-14
Publication date: 1996-05-23

Abstract

The invention relates to a process for releasing intracellular heterolog proteins in the Saccharomyces Cerevisiae yeast based on the osmotic sensitivity which confers the mutation slt2 at the temperature of 37 °C. Said osmotic sensitivity may be used for releasing the intracellular content either by change of temperature of the culture from 24° to 37° C, or by transfer of grown cells in an osmotically stabilized medium at said second temperature to a non-stabilized medium. In both cases, the cells undergo a lysis and release their intracellular content and, after centrifugation to eliminate the cellular rests, a preparation of proteins is obtained which is appropriate to initiate a process of purification of the protein of interest. This process results in a cost reduction since the use of equipment is avoided and the preparation of proteins which is obtained contains less impurities than that which is obtained through other processes.

Claims

MODIFIED CLAIMS [received by the International Office on April 15, 1996, June 15, 1996; original claims 1-5 replaced by modified claims 1-6 (2 pages)]

1.- Procedure for the release, in percentages higher than 50%, of heterologous proteins and other intracellular materials (such as VLP type particulate products) produced in Saccharomyces cerevisiae, based on the use of mutated strains in genes that affect cell integrity, stable under normal laboratory growth conditions (24-, simple nutritive media), and able to release the intracellular content by expression of the corresponding mutation under non-permissive conditions.

2. Process for the release of heterologous proteins produced in mutant strains of Saccharomyces cerevisiae according to claim 1, stable in the growth conditions suitable for the generation of a high cellular biomass.

3.- Procedure for the release of heterologous proteins from strains of

Saccharomyces cerevisiae according to claim 1 based on the osmotic sensitivity conferred on the cell mutations such as those affecting the SLT2 gene at non-permissive temperatures such as 37 ^* C, and which determine cell lysis of more than 50% of the cells in culture. .

4. Procedure for the release of heterologous proteins from strains of Saccharomyces cerevisiae according to claims 1 and 3, characterized in that the preparation of heterologous proteins and intracellular particulate products is carried out by any technique which, producing the expression of the corresponding mutation, leads to to the loss of cellular integrity due to the osmotic sensitivity of the cells.

5. Procedure for the release of heterologous proteins of strains of Saccharomyces cerevisiae according to claim 4, characterized in that the expression of the mutations can be caused in cells capable of expressing heterologous proteins or intracellular particulate products (ie transformed with the corresponding gene or genes) ) by thermal shock of cultures, ie elevation of the temperature to non-permissive levels in osmotically unprotected media, or - β

by osmotic shock of cells incubated at non-permissive temperature in osmotically protected medium.

6.- Procedure for obtaining intracellular heterologous proteins and particulate products characterized by the use of other yeast species other than Saccharomyces cerevisiae in autolytic mutants, affected in homologous genes of the SLT2 gene or in others whose mutation determines loss of cellular integrity, using an osmotic shock or a thermal shock.

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DECLARATION ACCORDING TO ARTICLE 19

The originality of the invention we believe is assured since the publication describes its use for obtaining heterologous proteins ("A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity." Alvarez et al. 1994. Journal of Biotechnology 5: 81-88) appeared sufficiently after the national patent application.

In the previous publication of our group ("Expression of mutations and protein safety by yeast conditional autolytic mutants in batch and continuous cultures", de la Fuente et al., 1993. Applied Microbiology and Biotechnology 38: 763-770), the release is described of homologous proteins as a part of a phenotypic study of the mutant strains, but its application to heterologous proteins is not proven at all, although the possibility of addressing this last development is noted in the discussion.

Therefore, the discovery of the procedure useful for releasing claimed heterologous proteins had not been previously published. In addition, when describing the aforementioned release of homologous proteins, osmotic shock was not used as a way to trigger cell lysis. Furthermore, the strains to be used for the heterologous release had to be improved to favor protein stability with respect to those described in the publication in Applied Microbiology and Biotechnology.

As regards the protein release procedure described in the publication "Isolation of recombinant proteins from Saccharomyces cerevisiae by use of osmotically fragüe mutant strains" (M. Broker, 1994. Biotechniques 16: 604-610), it can be said that it differs remarkably from the one claimed in our patent application. This difference affects in general to all the characteristics but we can fix especially in two. On the one hand, the latter (those described in Biotechniques 16: 604-610) are unstable strains whose growth is difficult since it only takes place in the presence of an osmotic stabilizer whatever the temperature of incubation (they are osmotic conditional strains). This contrasts with our strains (which are thermosensitive) that are perfectly maintained in culture at temperatures below 28 ° C, optimal for the growth of S.cerevisiae, without any special precautions and that can grow easily, therefore, in liquid medium until high biomass densities. On the other hand, said publication (Biotechniques 16: 604-610) ensures that with its procedure the release of more than 20% of the intracellular recombinant protein is never achieved by not using more than that percentage of cells, without the authors twenty

find an explanation for this phenomenon. This also contrasts with the intense release that is achieved with our procedure.

In view of the foregoing, we have modified the claims slightly and have added one, to better specify the specific, novel and differential characteristics of the claimed procedure.