BIOTECHNOLOGY TECHNIQUES Volume 9 No.6 (June 1995) pp.441 -444 Received as revised Is1 May

IWLEASE OF VIRUS-LIKE PARTICLES BY OSMOTIC SHOCK FROM A MUTANT STRAIN OF YEAST DEFICIENT IN CELI, INTEGRITY

Pablo Alvarez, Miguel Stichez, M&a Molina and CCsar Nomhela”

Departamento de Microbiologia II, l-‘acultad de Farmacia, Plaza de Ram6n y C@l, Univcrsidad Complulense, 2804&Madrid, Spain. Fax: 34 1 3941745.

SUMMARY A yeast system based on a S. cerevisiae mutant strain deficient in cell integrity, was used to

achieve the rclcasc of yeast expressed virus-like particles (vI.Ps) by simple osmotic shock. Yeast cells, grown in the prcscnce of IM sorbitol, lysed and rcleac;ed the intracellular content upon being transfcmzd to non-osmotically stahilizd medium. The release of VLPs was followcrl by Western blotting determination of the pl protein.

INTRODUCTION Yeast species are one of the host systems most widely used for the production of

helerologous proteins in industrial processes (Romanos et al., 1992). While much effort has hcen

made to obtain improved expression vectors and high yield strains, the isolation and downstream processing of intracellular proteins is still one of the main problems to be solved in an easy, low-cost

and less time consuming way. Although secretion is an alternative to cell disruption to achieve the release of proteins trl the

external medium from intacl cells, not all the recombinant products of interest are suitable for sccrclion. This is the case for virus-like particles (VLPs) that, hecausc of their large size (60nm), arc

produced only intraccllularly in S. cerevisiae yeast cells. The use of VI .Ps as a system for exprcssinn of foreign proteins in yeast has been rcccntly developed by Kingsman et a/.( 1991), and dcmonsrrated

as a powerful tool from a biotechnological viewpoint. Proteins fused to the structural protein subunit

pl of the Ty yeast retrotransposon arc assembled as non-infectious particles in which the protein of interest is on the surface. The ease of purification of the recombinant VI.Ps and their immunogenic

properties account for their multiple applications (for a review see Kingsman et u1..1991). In our laboratory, we have developed a new system for the relcasc of heterologous proteins produced

intracellularly from yeast, hased on a S. cerevisiae mutanl strain deficicnr in ccl1 wall inccgrity,

which lyses at 37‘j’c, in the absence of osmotic stabilizers. We have shown the ability of lhcsc

mutants lo retease about 70%; of the 2SklJ heterologous protein CAT (Chloramphenicol Acctyl Transfcrase) (Alvarez et al., 1994).

In this report we demonstrate thar not only relative small proteins can hc released hy lyt2 (~112) mutant cells, but also large complexes such as VI.Ps. in an efticient manner and without

appreciable degradation.

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MATERIALS AND METHODS Strains and Growth conditions

E. coli strain DH5a (recA1, e&Al, gyrA96, thil, hsdRI7, supE44, (rk-, mk+), relAI,FNIR, 1acZ MZS, F) was used for plasmid preparation. S. cerevisiae strain LDl is MATdMATrr Zyt2- l/lyt2-1, leu2-3.112/leu2-3.112, his4A14/his4A34.

Yeast cells were inoculated in synthetic medium lacking leucine (Sambrook et al., 1989), supplemented with 1M sorbitol, and grown at 37°C in an orbital incubator 250 rpm). Yeast transformation

Yeast cells were transformed with pMA5620 (Adams er al., 1987a) as described by Hinnen et al. (1978). Yeast strains containing recombinant plasmids are designated with the name of strain followed by the plasmid name, LDl/pMA5620. Flow cytometry assay

Details of flow cytometric measurements have been described previously (dc la Fucntc et al., 1992). Samples of 2-5~10~ cells were spun down, stained with 0.005% propidium iodide (PI) for 15’ at room temperature in the dark, spun down again, suspendend in lml phosphate buffered sahnc and analysed in a FACStar Pl.US flow cytometer (l&ton-Dickinson, San Jo&, CA, USA) equipped with an argon laser tuned to 488 nm (3OOmW). Gel electrophoresis and Western blotting analysis

Western blotting analysis was carried out as described by Towbin et al. (1979), using the Immun-Lite Assay Procedure (Rio-Rad). Following SDS-PAGE, proteins were transferred to the Immun-Lite membrane. Blots were probed with polyclonal anti-VLP antibody ( kindly supplied by Dra. Rosa Varona), at a 1:25000 dilution, and developed according to the instructions supplied with the kit.

Protein whole extracts were prepared as previously described (Mellor ef al., 1983). To prepare osmotic shock samples, cells were grown in shake flasks in synthetic medium lacking leucine plus 1M sorbitol at 37yC, centrifuged and resuspended in an equal volume of TE buffer pH 7.5, centrifuged again to separate cell ghosts and debris, and the supernatant loaded in the gel.

RESULTS AND DISCUSSION

The S. cerevisiue ljlt2 (~212) mutant strain LDl grew in liquid medium at 37QC only in the presence of an osmotic stabilizer, i.e. sorbitol (de la Fucntc er al. 1993). The expression of slt2 mutation at high tcmperaturc leads to the formation of an altered cell wall, which results in cell lysis

and protein release in hypotonic medium (i.e. buffer or distilled water) (Alvarez et al. 1994). We transformed S. cerevisiae LDl with the episomic expression vector pMA5620 (kindly supplied by

Dr. A. J. Kingsman) that carries a truncated S. cerevisiae TYA gene under the control of the high

level constitutive expression promoter of the yeast phosphoglycerate kinase (PGK) gcnc. The TYA

gene encodes the protein pl, which is analogous to retroviral gag proteins in that it aysernbles into virus-like particles (Ty-VLPs) that resemble rctrovirus cores (Mellor et al. 1985). Adams et al.

(1987b) have shown that overexpression of the truncated form of this gene leads to the intracellular

accumulation of large numbers of particles in yeast.

Western blotting analysis (Fig. 1) showed that strain LDl/pMA5620 produced VLPs, growing in a synthetic medium lacking leucine and supplemented with 1M sorbitol at 37gC. A band with the same electrophoretical mobility in SDS-PAGE as the pl protein from purified VLPs (lane

4) was de&ted in a cell extract obtain& by mechanical disruption (lane2). Other bands could also

be detected with the anti-p1 antibodies in this sample, probably due to aggregates in the c&se of bands above monomeric p 1, or degradation products of smaller size. Only a faint band corresponding to pl was observed in samples of culture medium after I6 hours of growth under thcsc conditions

(lane 5), confirming the intracellular production of the Vl,Ps. The small amount that could bc

detected in the osmotically stabilized extracellular medium was probably due to the release of VLPs from a small cell subpopulation (about 5%, Fig. 2A) of mutant cells that lyse at 37°C even in the

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Figure 1. Western blotting analysis of VLPs. Lane 1, total extract from LDl; lane 2, total

extract from LDllpMA5620; lane 3, protein size standards; lane 4, pure VLP, lane 5,

supernatant of LDllpMA5620 in osmotic stabilized medium; lane 6, supematant of

LDllpMA5620 after osmotic shock.

x.- A 20- B

Figure 2. Effect of osmotic shock on the viability of strain LDllpMA5620 growing at

37°C in 1M sorbitol stabilized medium (A) and resuspended in water @), determined by

means of flow cytometry. Fluorescence intensity is represented on the abcissa, in log units, and the relative number of cells is represented on the ordinate.

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presence of sorbitol. However, when the cells growing in stabilized medium were t.ransfcrred to TF buffer the release of a substantial amount of VLPs took place as shown by the intense pl protein band that could be observed by Western blotting analysis of the corresponding preparations (F&.1). Densitometer tracings of the fets indicated that the band of pl protein obtained after osmotic shock

(lane 6) was 11 times more intense than the one obtained from non-osmotically shocked cells (lane

3. The degree of cells lysis was followed by FACS analysis of cells stained with propidium

iodide (PI) as described by de la Fuente et al. (1992). Consistent with the detection of pl protein in

the external medium, WC observed that only a very small percentage (about 5%) (Fig. 2A) of the

cells were lysed in the sorbitol stabilized medium, whereas most of the cells had lost their integrity upon transfer to the non-stabilized solution (Fig. 2B).

Asenjo et al. (1993) proposed the use of lytic enzymes for the release of VLPs from yeast, but the system required a preparation of pure glucanase to avoid protein degradation and thus

necessitated a large scale production of the purified enzyme by developing a low-cost industrial

process. Western blotting analysis (Fig. 1) also showed that the present system, using the osmotic shock of LDl strain, lerl to the release of VLPs with significantly less degradation (lane 6) than

when obtained by mechanical breakage (lane 2 ). These results clearly indicate that the use of this mutant strain represents an easy, rapid and

efficient way to obtain large molecules as VLPs outside of yeast cells without further mechanical,

chemical or enzymatic treatment.

Acknowledgements

This investigation was supported by grants BIOT-(X90-165 (BKIDGE) from the European

Commission and PB91-919-01 from DIGICYT (Spain). We wish to thank A. Alvarez for the FACS

determinations, Dr. A. J. Kingsman for supplying plasmid pMA5620 and Dra. R. Varona for the

anti-VLP antibody.

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