ITMI942308A1 - METHOD OF RECOVERY OF BIOLOGICALLY ACTIVE PRODUCTS FROM MICROORGANISMS - Google Patents

Description

Description of the industrial invention entitled: "METHOD OF RECOVERY OF BIOLOGICALLY ACTIVE PRODUCTS FROM MICROORGANISMS"

The present invention relates to the release of recombinant or natural products by spore-forming bacteria (capable of forming spores), such as those of the genus Baciilus, during the sporulation process. Similarly, the present invention also relates to the release of such products, during the cell lysis process, by spore-forming bacteria rendered incapable of forming spores by genetic engineering techniques.

BACKGROUND OF THE INVENTION

Baci11us subtilis is widely used in genetic engineering. A large number of both prokaryotic and eukaryotic genes have been cloned and expressed in this member of the genus Bacillus (Simcnen N. &, Paiva I. Micrcbiol. Rev. 57: 109, 1993). This microorganism is mainly used for its ability to secrete proteins, but it has also been used for the endocellular expression of heterologous proteins (Velati Bellini A. et al., J. Biotechnol. 18: 177, 1991). One of the major problems encountered during the recovery of endocellular proteins from B. subtilis is the preparation of the lysate: in fact, the lysis can only occur through repeated passages in the French-Press (Velati Bellini A. et al. J. Biotechnol. 18: 177, 1991), enzymatic incubations (for example with lysoziroa; Penaiva M.A. & Salas M. Proc. Nati. Acad. Sci. USA 79: 5522, 1982), or prolonged sanitation (Prestidge L. et al. J. Bacteriol. 107 : 815, 1971). The interest in the development of a recovery system that does not use stressful conditions derives mainly from considerations about the integrity of the rewinding products. In fact, the preparation of the bacterial U sate is a truly critical step in the purification processes as it can influence the total quantity and biological activity of the recovered product as well as the macromolecular structure of the protein, its association with other cellular components and the presence of contaminants that can influence the subsequent purification steps. For this purpose, many studies have been carried out aimed at obtaining engineered bacterial strains capable of "releasing" receptive and healthy products. Culture media have been studied such as to make cell growth possible but in the same way to permeate the bacterial wall in order to allow a "passive release" of the recanbinant protein (Naglak T.J. & Wang H.J. Biotechnol. Bioengineer. 39: 732, 1992). It is known that a natural lysis process can occur in some bacteria. For example, when placed in unfavorable environmental conditions, B «subtilis generates endospars which are subsequently released by autolysis of the parent cell (Kaiser D. & Losick R. Celi. 73: 237, 1993; Shapiro L. Celi. 73: 841 , 1993; Partridge S.R. & Errington J. Molec. Microbics.

8: 945, 1993).

FIELD OF INVENTION

The present invention relates to the use of the natural process of sporulation (autolysis of the bacterial cell and formation of endDspore) in strains of spore-forming bacteria, for the recovery in intact and easily purifiable form of natural or re-swelling proteins produced by the bacterium, or of ncn molecules proteins derived from the bacterium itself. The present invention also relates to the recovery of the aforesaid molecules from the supernatant of genetically modified spore-forming bacteria so that they undergo autolysis without the formation of spores.

The field of the invention includes:

- the use of engineered strains of spore-forming bacteria for the recovery of re-swelling proteins from the sporulation supernatant;

- the use of strains of spore-forming bacteria for the recovery of natural proteins from the sporulation super atant;

- the use of spore-forming bacteria strains rendered non-spore-forming with genetic engineering techniques or other techniques, for the recovery of molecules produced by the bacterium from the autolysis supernatant;

- the use of the sporulation-germination system for the cyclic or semi-cyclic production of molecules produced by the bacterium. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the release and recovery of natural or recombinant proteins that are produced by microorganisms capable of forming endospores. More particularly, the invention relates to a process for the purification of embroidering or natural proteins from spore-forming bacteria which includes:

a) culture of spore-forming bacteria under conditions that favor the expression of the recanbinant or natural protein;

b) culture in a medium capable of inducing sporulation;

c) the isolation of the recombinant or natural protein from the sporulation supernatant.

In laboratory conditions, sporulation is obtained by making the microorganism grow in a "rich" medium, such as to support vegetative growth and the expression of the protein of interest (recanbinant or natural), and subsequently by transferring the bacteria to a medium suitable for inducing sporulation (for example, a medium that lacks essential factors for bacterial growth and / or that contains metals or other factors that favor the sporulation process, such as Difco Sporulating Medium; Jongman P.J., Perkins J.B., and Losich R., Proc. Nati. Acad. Sci. USA, 80: 2305, 1983). Growth and sporulation can also occur in the same medium (using the high cell density in culture as a signal for induction of sporulation) using for example the Potato Extract Medium (Johnson W.C. and Tipper D.J. J. Bacteriol. 146: 972, 1981). The first medium offers greater reproducibility, while the second is more suitable for cultivation in fermenters with quantitatively higher yields in biomass and protein. After sporulation, which involves autolysis of the bacteria, the culture medium contains the proteins that were present within the bacterial cell, including the protein of interest (natural or recanbinant).

The present invention also relates to the obtaining of the protein of interest (natural or recanbinant) from the culture medium, of bacteria engineered or otherwise modified in such a way that they are not able to complete the spore formation during the sporulation process (for example by deletion or inactivation of a factor essential to the completion of the sporulation process, such as one of the spore proteins). In this way the bacterium that receives the sporulation signal (high density in culture, lack of adequate nutritional elements, particular signals of induction of sporulation) begins the sporulation process and undergoes autolysis, releasing the protein of interest into the supernatant, without however, be able to form the spore.

The present invention also describes a semi-continuous production system, which exploits the characteristics of resistance and stability of the spores. In this system, the spores obtained in the first part of the process are put to germinate in a fresh nutrient medium such as to restore bacterial growth and the production of the whole protein (natural or recalcitrant). The release of the protein is obtained at the time of the subsequent sporulation of the microorganism.

The present invention includes the recovery from the autolysis / sporulation supernatant of both natural proteins produced by the bacteria (for example some toxins having insecticidal activity) and of rich proteins produced by bacteria engineered with the genes coding for them.

In the present invention, three processes are described by way of healthy example: a releasing system of a recanbinant protein (the reeectoral antagonist of IL-10: IL-Ira; Dinarello C.A. & Ihonpson R.C. lim itici. Today 11: 404, 1991) ; the purification of the recombinant protein itself, a cyclical system of use of the spores.

DESCRIPTION OF THE FIGURE

Figure 1: Densitometric analysis after separation on SDS-PAGE of IL-lra in B. subtiiis preparations. The electrophoretic migration of recurrent IL-1ra is indicated by an arrow. The molecular weights of the standards are reported in KD.

panel: densitometry of molecular weight standards (Bio Rad); panel: densitometry of the cellular iisate supernatant (Se),

15 μl.

panel: densitometry of the fraction enriched in IL-lra from Sc,

15 μl;

panel 4: densitometry of the sporulation supernatant (Ss), 15 μl;

panel 5: densitometry of the fraction enriched in IL-lra by Ss,

15 μl.

EXAMPLES EXAMPLE 1: Cionage and expression of a recombinant protein (IL-lra) in a B. subtiiis strain

The cDNA encoding the IL-1ra protein is for example isolated by PCR from a pool of cDNA, prepared with classical methods from cells of monocyte-macrophage origin. The oìigonucieotides usable for the selective amplification of the cDNA encoding IL-lra are indicated:

The forward oliganucleotide was designed so as to insert the EcoRI restriction site immediately upstream of the codon encoding the amino acid Methionine (indispensable for directing the initiation of mRNA translation), followed by the codon encoding the first amino acid of the mature form of the protein (R26). Similarly, through the reverse oligonucleotide a Pst1 restriction site is inserted immediately downstream of the stop codon of the protein. The amplified fragment is cloned in the EcoRI and Pstl sites of the pSM671 expression vector, obtaining the pSM441 plasmid. The piasmide pSM441 is transferred into cells of the SMS118 strain of subtilis (leu-, pyrDI-, npr-, apr-) by standard methods. A culture of the aforementioned transformed bacteria can be stored at -80 ° in 15% glycerol until use.

EXAMPLE 2: Growth of the engineered B. subtilis strain and release of the recombinant protein

Two 100ml aliquots of LB medium containing 5 mg / 1 of chlorampheniool are inoculated with a 1: 200 dilution of a primary inoculum prepared in the same medium from a glycerol stock at -80 ° C. The culture is grown under stirring at 37 ° C for 7 hours. Bacteria are collected by centrifugation (3,000 x g, 20 ', 4 ° C), washed in 50 mM pH 6.25 MES-NaCH tarrpone containing 1 nM ED1A, and frozen at -80eC. For the preparation of the cell lysate, the bacteria are thawed and resuspended in 10 ml of tarrpone MES-NaCH 25

rtM pH 6.25 containing 1 nM EDTA (Buffer A) and used using LUI senioatore XL-2020 (Heat Systems) with an output power of 80 W for 14 cycles of 30 "scnreaction and 30" cooling in a bath of ice water. The cellular U sate is centrifuged at 30.OOOxg for 30 'at 4 ° C, the supernatant (Se) is recovered, avoiding to collect the floating layer on top of the centrifuge tube, and analyzed on SDS-polyacrylamide gel. For the preparation of the sporulation supernatant, the bacteria of the second aliquot are collected aseptically by centrifugation (3,000 x g, 30 'at 4 ° C) and resuspended in 10 ml of "Difco Sporulating Medium" with or without chloramphenicol. The incubation is continued overnight at 35 ° C with agitation. The spores are collected by centrifugation (3,000 x g, 30 'at 4 ° C) and the supernatant (Ss) is analyzed on SDS-polyacrylamide gel. The reciprocating protein is present in comparable quantities in both preparations (Figure 1 and Table 1).

EXAMPLE 3: Purification of the recombinant protein produced by the engineered strain of B. subtilis

Se and Ss, kept frozen at -80 ° C until ready for use, are thawed and filtered through a Millex HV 0.45 μm filter (Millipore). For Ss, the pH is adjusted to 6.25 with HCl. The two supernatants are incubated for 3 hours at 4 ° C under slow shaking, with 1 ml of Q-Sepharose Fast Flow (Pharmacia), or other balanced anion exchanger, in buffer A, using a disposable Poly-Prep column (Bio- Rad). The non-adsorbed material is collected and incubated under the same conditions with 1 ml of S-Sepharose Fast Flow (Pharmacia), or other cation exchanger, previously equilibrated with tanpcne A. After washing with buffer A, the resin is eluted with 2 ml of buffer A containing 0.5 M Nad. The eluate is concentrated to 0.25 ml using, for example, the Centriccn 10 (Amiccn) concentration system by centrifugation.

EXAMPLE 4: Electrophonetic analysis on SDS-polyacrylate gel Carpioni of protein are analyzed on a 13.5% acrylantnide gel according to the Laemmly method and stained with Coomassie R-250. The densitcmetric analysis is performed with a laser densitometer (Molecular Dynamics Personal Densitometer) and the data evaluated with the use of the Image Quant (Molecular Dynamics) software. Figure 1 shows the result of the partial purification: the total protein content of the preparation from Ss is lower, but with a higher degree of purity, compared to the preparation from Sc (see also Table 1).

EXAMPLE 5: Protein determination

Protein determination is performed using the "Bio-Rad Protein Assay" kit according to the supplier's specifications. BSA (Bio-Rad protein standard I) is used as a standard for the determination of proteins in the starting carpions, while an IL-lra standard is used for the analysis of the proteins of the purified fractions. The standard IL-1ra concentration is calculated by measuring the 0.1% A280 of the protein solution in a 6 M guanidine hydrochloride solution prepared in 20 nM phosphate buffer pH 6.5. Under these conditions the 0.1% of IL-lra, calculated with the use of the PC / GENE software (IntelliGenetics) is 0.910. The results of the determinations are reported in Table 1.

EXAMPLE 6: Biological activity of IL-lra

The inhibitory activity of IL-lra is measured in the proliferation test of thymocytes sights. Thymodts (6 x 105 cells / well in 96-well Costar plates) of 5-7 week old C3H / H6J {Jackson Labs) mice cultured in RPMI-1640 (Gibco) with 5% fetid bovine serum (FBS, Hyclcne ), β -mercap t o-e tanol 25 μΜ, 50 μg / ml of gentamicin (Sigma) and 1.5 μg / ml of phytohemagglutinin (PHA, Wellcome). A fixed dose of hurIL-1B (30 pg / ral) alone or in the presence of different doses of standard or purified IL-1ra (from 10 pg / ral to 100 ng / ml) is added, in triplicate, to the wells. The plate is incubated at 37 ° C in humid air with 5% CO2 for 72 hours and subsequently incubated overnight with 0.5 μCi of 3H TdR (NEN). Cells are collected on glass fiber filters and H TdR incorporation levels determined with a Pharmacia Betaplate scintillatole. One unit of IL-1ra is calculated as the amount that inhibits 50% of the proliferation induced by IL-1. From the data reported in Table 1 it is evident that there are no significant differences in activity between the two preparations.

EXAMPLE 7: Semi-cyclic system for the production of proteins and spores For the growth of the microorganism (vegetative phase-sporogenic phase), 100 ml of LB medium containing 5 mg / 1 of chloramphenicol are inoculated with a 1: 200 dilution of an inoculum primary prepared in the same medium starting from a glycerol stock at -80 ° C. The culture is grown under stirring at 37 ° C for 7 hours. The bacteria are collected aseptically by centrifugation (3,000 x g, 30 'at 4 ° C) and resuspended in 10 ml of "Difoo Sporulating Medium" without chloramphenicol. The incubation is continued for one night at 35 ° C under agitation. The spores are separated from the supernatant by centrifugation (3,000 x g, 30 'at 4 ° C). The supenatant is used for protein purification as described in Example 3. The collected spores are used as inoculum in nutrient medium (LB) containing chloramphenicol 5 mg / 1. In this soil they are induced to germinate and re-enter the vegetative cycle. After 7 hours from the initial moment of growth, the cells are again aseptically collected and transferred to "Difco Sporulating Medium" where they are again induced to sporulate.

Claims (7)

CLAIMS 1. Process for the purification of recombinant or natural proteins from spore-forming bacteria which includes: a) culture of sporogenic bacteria under conditions that favor the expression of the inant or natural re-edited protein; b) culture in a medium capable of inducing sporulation; c) the isolation of the inant or natural blackmail protein of the sporulation supem atant. 2. Process according to claim 1 which further comprises the separation of the spores from stage b) and their reuse, after returning to the germinative stage, in the culture of stage a). 3. Process according to claim 1 and / or 2 in which the sporogenic bacteria sinus strains of Bacillus subtilis. Process according to any one of claims 1-3 for the purification of recombinant proteins from engineered sporogenic bacteria. Process according to any one of the preceding claims in which the recombinant protein is the receptor antagonist of IL-Ιβ (IL-1ra). Process according to any one of claims 1-3 wherein the sporogenic bacteria are not engineered and the protein of interest is a natural product of the bacterium. 7. Process according to any one of the preceding claims in which the sporogenic bacteria have been rendered unable to form the spore, by genetic engineering or other suitable techniques, to use the autolysis step without spore formation for recovery of the proteins of interest.