LT7068B - Cell culture method - Google Patents

Abstract

The invention provides a method for producing a clarified cell culture comprising a recombinant protein, wherein the method comprises: i) inoculating a bioreactor with a host cell to provide a cell culture, wherein said host cell is an Escherichia coli transformed with an expression system comprising: a) a nucleic acid encoding a recombinant protein operably linked to a first inducible promotor; and b) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding PelB secretory signal peptide operably linked to a second inducible promotor, ii) fermenting the cell culture under conditions that: a) allow the expression of the recombinant protein; and b) allow permeabilization of the cell so as to form a spheroplast that secretes the recombinant protein; iii) clarifying the fermented cell culture comprising the secreted recombinant protein, wherein said clarifying comprises a primary clarification step using a first clarification filter having a pore size that provides a retention range of at least about 0.4 μm or more, wherein the clarification step does not comprise centrifugation.

Description

TECHNICAL FIELD

The present invention relates to a system for expressing recombinant proteins using E. coli.

STATE OF THE ART

Escherichia coli is a frequently chosen microorganism for the production of recombinant proteins. The main reasons for this popularity are the high growth rates and resolution levels, as well as the simple and inexpensive culture media required. However, E. coli is a poor protein secreter, and methods for intracellular protein production using E. coli require cell disruption and removal of cellular debris. In contrast, cellular production of recombinant proteins and enzymes greatly reduces the complexity of the bioprocess production method and improves the quality of the recombinant product, while simplifying protein detection and purification. Furthermore, an environment free of cell-associated proteolytic degradation has been shown to provide an optimal environment for the formation of protein spatial structures.

Various methods can be used to facilitate the production of intracellular proteins in E. coli. Typically, the initial steps of these methods involve lysing or disrupting bacterial cells to disrupt the bacterial cell wall and release cellular proteins into the extracellular environment. After this release, the required proteins are purified from the extracts, usually by several chromatographic steps.

Several methods have been shown to be useful for the release of intracellular proteins from bacterial cells. These include the use of chemical lysis, physical disruption methods, or a combination of chemical and physical methods (Felix, H., Anal. Biochem. 120:211-234 (1982)).

Chemical methods of disrupting bacterial cell walls that have proven useful include treating the cells with organic solvents such as toluene (Putnam, S.L., and Koch, A.L., Anal. Biochem. 63:350-360 (1975); Laurent, S.J., and Vannier , F. S., Biochimie 59:747-750 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982)) by chaotropes such as guanidine salts (Hettwer, D., Biotechnol. Bioeng. 33:886-895 (1989)), antibiotics such as polymyxin B (Schupp, J.M., BioTechniques 19:18-20 (1995); Felix, H., Anal. Biochem. 120: 211-234 (1982)), or enzymes such as lysozyme or lysostaphin (McHenty, C.S., and Kornberg, A., J. Biol. Chem. 252(18):6478-6484 (1977); Cull, M., and McHenry, Meth. 182:147-153; J. Cell Suppl. 016 (1992); ., et al., in Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), pp. 17-38; Ausubel, F.M., et al., in Current Protocols in Molecular Biology, New York: John Wiley & Sons (1993), pp. 4.4.1-4.47). The effects of these various chemicals can be enhanced by simultaneously treating the bacterial cells with detergents such as Triton X-100®, sodium dodecyl sulfate (SDS) or Brij 35 (Laurent, S.J., and Vannier, F.S., Biochimie 59:747-750 (1977) ; Felix, H., Biochem. 120:211234; Hettwer, H., Bioeng., 33:886-895; , Meth. Enzymol. 182:147-153 (1990); Schupp, J.M., et al., BioTechniques 19:18-20 (1995)) or proteins such as bovine serum albumin or spermidine (McHenry, C.H. and Komberg, A. Biol. 252(1977): 120:211-234, Hughes, J., et al ., J. Cell Biochem. Suppl. 0 16 (Part B):84 (1992)).

In addition to these various chemical treatments, several physical degradation methods have been used. These physical methods include osmotic shock, such as suspension of cells in a hypotonic solution in the presence or absence of emulsifiers (Roberts, J.D., and Lieberman, M.W., Biochemistry 18:4499-4505 (1979); Felix, H., Anal. Biochem. 120 :211-234 (1982)), drying (Mowshowitz, D.B., Anal. Biochem. 70:94-99 (1976)), mixing with granules such as ball milling (Felix, H., Anal. Biochem. 120: 211234 (1982); Cull, M., and McHenry, Meth. Enzymol. 182:182:147-153 (1990)), e.g. freeze-thaw cycles (Lazzarini, R. A., Nature New Biol. 243:17-20 (1975); Felix, H., Biochem. 120:211-234 (1982)), sonication (Amos, H., et al., J. Bacteriol. 94: 232-240 (1967); Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York, John Wiley & Sons (1993), pp. 4.4.1-4.47) using a French press pressure chamber (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York, John Wiley & Sons (1993), pp. 16.8.6-16.8.8). Other methods combine these chemical and physical disruption methods, such as lysozyme treatment followed by sonication or pressure to maximize cell disruption and protein release (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York , John Wiley & Sons (1993), pp. 4.4.1 -4.47).

These disruption methods have several advantages, including their ability to rapidly and completely disrupt bacterial cells in a way that maximizes the release of intracellular proteins.

However, these methods also have distinct drawbacks. For example, physical methods essentially involve tearing and breaking down bacterial cell walls and plasma membranes. These processes result in extracts that contain many solids, such as membrane or cell wall fragments, which must be removed from the extracts by conventional centrifugation before the recombinant proteins can be purified. This need for centrifugation limits the size of the batch that can be processed at one time to the volume of the available centrifuge; thus, large-scale production preparations are impractical, if not impossible. In addition, physical methods and many chemical methods usually lead to the release of not only desired intracellular proteins, but also unwanted nucleic acids and membrane lipids from cells (the latter especially occur with organic solvents). These unwanted cellular components also complicate subsequent purification processes of desired proteins by increasing the viscosity of the extracts (Sambrook, J., et al., in: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), pp 17-38; Cull, C.S., Meth. Enzymol. 182:147-153 (1990)) and binds with high avidity and affinity to nucleic acid modifying proteins such as DNA polymerases. RNA polymerases and restriction enzymes.

Naturally occurring lytic and symbiotic bacteriophages have the ability to induce host cell lysis by expressing specific proteins during the lytic cycle. These lytic proteins have been identified and extensively studied in, for example, T4 and T7 bacteriophages. Lysing proteins include both holins and lysozymes. Cholins form stable and non-specific lesions in the cytoplasmic membrane, which allow lysozymes to enter the peptidoglycan layer. Lysozymes exhibit muralis activity against three different types of covalent bonds (glycosidic, amide and peptide) in the peptidoglycan polymer of the cell wall. Together, the activity of choline and lysozyme disrupts the two cell membranes of gram-negative bacteria, thereby causing cell lysis.

E. coli was engineered to express T4 bacteriophage lysis proteins to provide programmed cell lysis to improve the efficiency and cost-effectiveness of downstream processing for recombinant protein production (M. Morita, K. Asami, Y. Tanji, and H. Unno, "Programmed Escherichia coli Cell Lysis by Expression of Cloned T4 Phage Lysis Genes," Biotechnology Progress , vol. 3, pp. 573-576, 2001).

Escherichia coli-based constructs are the gold standard for the production of recombinant proteins where post-translational modifications are unnecessary. The main reasons for the popularity of E. coli are the high growth rates and expression levels, as well as the simple and inexpensive culture media.

However, E. coli is not a perfect host because it does not normally secrete proteins into the extracellular medium. Thus, one problem with methods using E. coli is that the cell disruption step becomes limiting and is not cost-effective on a pilot or industrial scale when implementing a closed and/or continuous process.

One problem with these methods is that the resulting recombinant proteins usually contain impurities. This is, for example, a problem in the production of nucleic acid-modifying enzymes, as enzyme preparations are typically contaminated with nucleic acids (eg, RNA and DNA). This impurity nucleic acid can come not only from the organisms that are the source of the enzyme, but also from unknown organisms present in the reagents and materials used to purify the enzyme after it is released from the cells. This is particularly true for reverse transcriptase or DNA polymerase enzymes, as they are commonly used in nucleic acid molecule amplification and synthesis methods (eg polymerase chain reaction (PCR), especially RT-PCR). In such a sample, the presence of contaminating DNA or RNA in enzyme preparations is a major problem, as it can lead to false amplification or synthesis results. In addition, with the growing demand for enzymes as biocatalysts in pharmaceuticals, additional requirements are placed on the quality of enzyme preparations, such as specificity, consistency when passing from one series to another, the absence of impurities of animal origin and antibiotics, which must necessarily meet regulatory requirements.

Thus, there remains a need for methods of producing recombinant proteins comprising E. coli that are substantially free of impurities such as nucleic acids. These methods can eliminate the risk of contamination, for example from the environment and personnel, and enable a faster and parallel production process in a single-process facility.

Thus, there remains a need for methods of protein preparation that are more efficient, cost-effective (for example, on pilot and industrial scale), and substantially free of unwanted cellular components such as nucleic acids.

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention, there is provided a method for producing a clarified cell culture comprising a recombinant protein, wherein the method comprises:

(i) inoculating the bioreactor with a host cell to obtain a cell culture, wherein said host cell is Escherichia coli, a transformed expression system comprising:

(a) a nucleic acid encoding a recombinant protein operably linked to a first inducible promoter, wherein the protein is vicronectin (VTN); and

b) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter;

(ii) cell culture fermentation under conditions which:

(a) enables expression of the recombinant polypeptide; and

(b) enables cell penetration to form a spheroplast that secretes the recombinant polypeptide;

iii) purifying the fermented cell culture comprising the secreted recombinant protein, wherein said purification comprises

(a) a nucleic acid inactivation step prior to the primary purification step, comprising adding a nucleic acid inactivating agent to the cell culture, wherein optionally the nucleic acid inactivating agent is benzonase; and

(b) a primary clarification step using a first clarification filter having a pore size of at least about 0.4 μm or greater retention limits, where the clarification step does not include centrifugation.

Conveniently, in the methods of the present invention, the method further comprises a secondary clarification step using a second filter having a pore size of at least 0.4 μm or greater retention limits.

Conveniently, in the methods of the present invention, the primary clarification step uses a first filter having a pore size that provides retention limits of at least 1.0 to 3.0 μm.

Suitably, in the methods of the present invention, the secondary clarification step includes a second filter that provides a pore size of at least about 0.22 μm, at least about 1.0 to about 3.0 μm, or at least about 0.4 to about 0.8 μm retention limits.

Suitably, in the methods of the present invention, the primary clarification step and/or the secondary clarification step is microfiltration.

Suitably, in the methods of the present invention, microfiltration is depth filtration.

Suitably, in the methods of the present invention, microfiltration is selected from normal flow filtration (NFF) or tangential flow filtration (TFF).

Suitably, in the methods of the present invention, the clarified cell culture has a turbidity of less than about 20 NTU.

Conveniently, in the methods of the present invention, said fermentation step comprises adding a stabilizing agent to the cell culture.

Conveniently, in the methods of the present invention, the process is carried out in a closed system such that a sterile flow is maintained between the bioreactor and the first and second clarification filters.

Conveniently, in the methods of the present invention, the process also includes the step of subjecting said clarified cell culture to one or more of the purification steps of said recombinant protein.

The invention also provides a method of producing a protein comprising:

(i) culturing a recombinant Escherichia coli cell under conditions which:

(a) enables expression of the protein; and

(b) enables cell penetration to form a spheroplast that secretes the protein; and (ii) isolating the protein from the culture without complete lysis of the recombinant cell, wherein the cell comprises an expression system comprising:

(a) a nucleic acid encoding a recombinant protein operably linked to a first inducible promoter, wherein the protein is vicronectin (VTN); and

(b) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter.

Suitably, the methods of the invention do not include: (i) mechanical cell disruption; (ii) addition of an exogenous enzyme that degrades the cell wall; and (iii) transforming the cell with expression vectors containing a nucleic acid encoding an exogenous enzyme that disrupts the cytoplasmic membrane.

Conveniently, in the methods of the present invention, the nucleic acid encoding the protein operably linked to the first inducible promoter is present in the first construct, and the nucleic acid encoding T4 lysozyme in frame with the nucleic acid encoding the PelB secretory signal peptide is present in the second construct.

Conveniently, in the methods of the present invention, the first inducible promoter and the second inducible promoter are induced sequentially.

Conveniently, in the methods of the present invention, the first inducible promoter and the second inducible promoter are simultaneously induced.

Suitably, in the methods of the present invention, the first inducible promoter is an IPTG-inducible promoter, optionally wherein the IPTG-inducible promoter is PT7.

Suitably, in the methods of the present invention, the second inducible promoter is an arabinose-inducible promoter, wherein optionally the arabinose-inducible promoter is araB.

Conveniently, in the methods of the present invention, the T4 lysozyme enzyme is encoded by the gene E.

Suitably, in the methods of the present invention, the E. coli is Escherichia coli B strain.

Suitably, in the methods of the present invention, the fermentation or culturing comprises changing the temperature from a first temperature to a second temperature, optionally wherein the second temperature is lower than the first temperature.

The invention also provides a recombinant protein produced using the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described below with reference to the accompanying drawings, in which:

plasmid structures are shown in the figure: (A) pET29-optVTN, (B) T4 gene gpe expression vector pACYC184-PT7-pelB-gpe-lacI.

the figure shows a microscopic view of E. coli cells before and after induction of gpe gene expression.

the figure shows the transformation of two different strains of E. coli with target plasmids. (A) Culture growth when JS007 E. coli strain and BL21 (DE3) E. coli strain were transformed with target plasmids. Induction of expression with 0.1 mM IPTG (OD600 1.0-1.5) is indicated in the graph. After 7 hours of incubation, the BL21 (DE3) culture had an optical density of 12.40, whereas the JS007 culture had an optical density of 6.60. (B) VTN protein expression result of JS007 strain (left) and BL21 (DE3) strain (right). References: (1), (6) - Page Ruler Prestained Protein Ladder (Thermo Scientific); (2), (7) - total protein fraction before induction; (3), (8) - total protein fraction after induction (0.1 mM IPTG, 4.5 h at 25 °C); (4), (9) - soluble protein fraction after induction; (5), (10) - insoluble protein fraction after induction. Target proteins are indicated by arrows.

the figure shows a comparison of scale-up fermentation, Biostat A, Biostat C and SUF. (A) Comparison of biomass and medium fractions for fermentation at different scales. References: (5), (10), - Page Ruler Prestained Protein Ladder (Thermo Scientific); (1) - biomass, biostat A before induction; (2) - biomass, flask after induction; (3) - biomass, biostat A after induction; (4) - biomass SUF, after induction; (6) - medium, biostat A before induction; (7) - medium, flask after induction; (8) - medium, biostat A after induction; (9) - medium, SUF after induction; SDS PAGE gel 4-12% Bolt. (B) Typical profile of the fermentation process in Biostat A.

the figure shows the reproducibility of the VTN fermentation process in a large-scale 300 L SUF. (A) Fermentation profiles of the three fermentations. (B) SDS PAGE analysis of supernatant fractions after the fermentation process for individual fermentations. References: (1) - Page Ruler Prestained Protein Ladder (Thermo Scientific); (2), (3), (4) supernatant fraction after autolysis of different fermentations in 300 l SUF. 4-12% Bolt SDS gel.

figure shows a schematic of the purification process used to obtain various recombinant proteins such as VTN (vitronectin).

DETAILED DESCRIPTION

The reader's attention is directed to all articles and documents that are filed with or prior to this specification relating to this application and that are open to the public in connection with this specification, and the contents of all such articles and documents are incorporated herein by reference.

Accordingly, the invention provides a method for producing a purified cell culture comprising a recombinant protein, wherein the method comprises:

(i) inoculating the bioreactor with a host cell to obtain a cell culture, wherein said host cell is Escherichia coli, a transformed expression system comprising:

(a) a nucleic acid encoding a recombinant protein operably linked to a first inducible promoter, wherein the protein is vicronectin (VTN); and

b) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter;

(ii) cell culture fermentation under conditions which:

(a) enables expression of the recombinant polypeptide; and

(b) enables cell penetration to form a spheroplast that secretes the recombinant polypeptide;

iii) purifying the fermented cell culture comprising the secreted recombinant protein, wherein said purification comprises

(a) a nucleic acid inactivation step prior to the primary purification step, comprising adding a nucleic acid inactivating agent to the cell culture, wherein optionally the nucleic acid inactivating agent is benzonase; and

(b) a primary clarification step using a first clarification filter having a pore size of at least about 0.4 μm or greater retention limits, where the clarification step does not include centrifugation.

As an advantage, the inventors have found that recombinant E. coli spheroplasts secrete the protein prior to cell lysis.

The inventors have shown that the coordinated expression of a nucleic acid encoding T4 lysozyme linked to a pectate lyase B (PelB) secretion signal sequence together with a nucleic acid encoding a recombinant polypeptide (eg, vitronectin) provides a highly efficient way to express large amounts of recombinant protein, e.g. vitronectin, without the need to harvest the recombinant cells from the fermentation culture and subsequently lyse the cells mechanically or enzymatically to release the recombinant protein. In the event that the T4 phage lysozyme gene is cloned under a tightly controlled promoter such as araB, periplasmic accumulation of T4 phage lysozyme can be induced by the addition of an inducer (eg, arabinose) during the appropriate fermentation process. Expression of T4 phage lysozyme with a PelB signal sequence facilitates translocation of T4 lysozyme to the periplasmic region, where the enzyme degrades the peptidoglycan layer, leading to morphological changes in the cell leading to spheroplast formation.

A recombinant polypeptide gene, such as the vitronectin gene, is cloned under the control of an inducible promoter, such as PT7 or lacUV5, and expression of the protein can be induced by adding an inducer, such as IPTG, during the appropriate fermentation process. By controlling nucleic acid expression of a recombinant polypeptide such as vitronectin and T4 phage lysozyme by separate promoters, their expression can independently regulate the production of each protein during fermentation. The inventors have shown that spheroplasts induced by T4 lysozyme secrete a recombinant protein such as vitronectin into the culture supernatant in the absence of a secretion signal sequence. The inventors have observed that recombinant expression of a recombinant protein such as vitronectin has no deleterious effect on the host cell. In fact, the inventors demonstrated that induced spheroplasts were able to de novo express a recombinant protein such as vitronectin as little as 4 hours after T4 lysozyme induction.

Furthermore, the inventors have shown that the resulting fermented cell culture can be successfully clarified using a clarification filter having an unusually large pore size that provides retention limits of at least about 0.4 μm. This is completely unexpected, as the established primary purification methods for E. coli rely on tangential flow filtration using a <0.2 μm pore size filter combined with centrifugation to remove cell debris.

Furthermore, the inventors have shown that fermented spheroplast cell cultures can be efficiently clarified without the use of centrifugation. By eliminating the centrifugation step, fermentation and clarification can be carried out in a closed system, eliminating the risk of external contamination.

Thus, in E. coli, a transformed expression system that enables cell permeability, in combination with a clarification process that does not require centrifugation, not only reduces the risk of contamination during protein production, but additionally eliminates a number of downstream processing methods in the production stages, including: cell harvesting, mechanical and/or enzymatic cell lysis, cell debris separation, nucleic acid precipitation followed by separation by filtration or centrifugation. Thus, defined methods provide high protein yields at high cell densities and scale-up. In addition, eliminating the need to lyse host cells and associated centrifugation steps reduces large-scale processing times compared to conventional lysis-based methods.

Resolution system

A method of the invention utilizes an expression system that comprises i) a nucleic acid encoding a recombinant protein operably linked to a first inducible promoter; and ii) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter, wherein the protein is vitronectin (VTN).

As used herein, the term "vitronectin" or "VTN" refers to a 75 kD glycoprotein that is reported to bind to a variety of biological ligands and play an important role in tissue remodeling. Vitronectin may be of human origin and may have the sequence defined in NCBI Sequence ID: AAH05046.1. Accordingly, vitronectin may comprise the sequence of SEQ ID NO. 1.

As used herein, the term "T4 lysozyme," "E protein," or "gpe" refers to a cytoplasmic muramidase that facilitates lysis of T4 phage-infected bacterial cells, thereby releasing replicated phage particles (Tsugita and Inouye, J. Mol. Biol., 37: 20112 (1968); Tsugita and Inouye, J. Biol. Chem., 243: 391-97 (1968), Uniprot accession: D9IEF7), or a mutant, derivative or fragment having DNA polymerase activity. It is encoded by the T4 bacteriophage gene and hydrolyzes the bonds between N-acetylglucosamine and N-acetylmuramic acid residues in the rigid peptidoglycan layer of the E. coli cell envelope. The enzyme is a single polypeptide chain with a molecular weight of 18.3 kDa. It is approximately 250-fold more active than HEW-lysozyme on bacterial peptidoglycan (Matthews et al., J. Mol. Biol., 147: 545-558 (1981)). The optimal pH value for T4 lysozyme enzyme activity is 7.3, and for HEW-lysozyme - 9 (The Worthington Manual; pp 219-221). The T4 enzyme may have the sequence defined in NCBI reference sequence: NP_049736.1. The T4 enzyme may have the sequence of SEQ ID NO: 2.

As used herein, the term "recombinant" refers to proteins that are engineered, prepared, expressed, engineered, produced and/or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell. In some embodiments, the recombinant protein is a functional variant derived from a polypeptide of SEQ ID NO. 1. For example, a variant may be a functional variant of vitronectin. In some embodiments, the polypeptide sequence of the functional variant is at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, identical to SEQ ID NO. 1. The percentage of identity between two sequences depends on the number of identical sequence positions, taking into account the number of gaps and the length of each gap that must be introduced to optimally align the two sequences. Comparison of sequences and determination of the percentage identity between two sequences can be performed using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the (Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm that was incorporated into the GAP program in the GCG software package (available at http: //www.gcg.com) using the BLOSUM 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters is particularly preferred (and one that should be used if one of ordinary skill in the art is unsure what parameters should be applied to determine whether a molecule satisfies the sequence of the invention in identity or homology constraints) is a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap expansion penalty of 4, and a frameshift gap penalty of 5. Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using an algorithm (Meyers et al. (1989) CABIOS 4:11-17)) which was entered into the ALIGN program (version 2.0) using the PAM120 weight residue table, a gap length penalty of 12 and a spacing penalty of 4.

Expression systems for use in the method of the present invention can be formed using methods well known in the art. In general, a cDNA or genomic DNA encoding a recombinant protein such as vitronectin or T4 lysozyme is suitably inserted into a replicable vector for expression in E. coli cells under the control of an E. coli appropriate promoter. Suitable vectors are well known in the art and the selection of an appropriate vector will depend on the size of the nucleic acid to be inserted into the vector and the host cell to be transformed by the vector. Each vector has different components. For example, bacterial transformation vectors will include a recombinant polypeptide signal sequence as well as a recombinant polypeptide promoter. Preferably, they include the origin of replication and one or more marker genes.

Preferably, the vectors are derived from species compatible with the host cell and are used in conjunction with bacterial hosts, i.e. i.e. vector compatible with E. coli. Preferably, the vector contains a site of replication and markers to ensure phenotypic selection of the transformed cells. Expression vectors for use in the present invention preferably have a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Such sequences are well known for various bacteria.

Expression vectors for use in the present invention preferably contain a selectable gene or a selectable marker. This gene encodes a protein essential for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed by the vector but carrying the selection gene do not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply important nutrients not present in the complex medium.

Expression vectors for use in the present invention contain an inducible promoter that is recognized by the host bacterial organism and that is operably linked to a nucleic acid encoding a polypeptide of interest, ie. i.e. recombinant protein such as vitronectin or T4 lysozyme.

Inducible promoters suitable for use with bacterial hosts such as E. coli include the β-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979 )), the arabinose promoter system, including the araBAD promoter (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992); Guzman et al., J. Bacteriol., 177: 4121-4130 (1995); Siegele and Hu, Natl. Sci. USA, 94: 8168-8172 (Haldimann et al., 180: 1277-1286) tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980) and EP 36,776), PLtetO-1 and Plac/ara-1 promoter (Lutz and Bujard, Nucleic Acids Res., 25: 1203-1210 (1997)) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)).

As used herein, the term "inducible promoter" refers to a promoter that, upon binding to a transcription factor, directs transcription at an increased or decreased rate.

As used herein, the term "transcription factor" refers to any factor that can bind to the regulatory or control region of a promoter and thereby affect transcription. Transcription factor synthesis or promoter binding ability in a host cell can be controlled by treating the host with an "inducer" or by removing a "repressor" from the host cell's environment. Accordingly, to regulate the expression of an inducible promoter, an inducer is added to or a repressor is removed from the culture medium of the host cell.

As used herein, the phrase "causing expression" or "inducing expression" refers to increasing transcription from specific genes by acting on cells containing such genes as an effector or inducer.

As used herein, the term "inducer" is a chemical or physical agent that, when introduced into cells, increases the amount of transcription from specific genes. These may be small molecules whose effects are specific to particular operons or groups of genes and may include sugar, alcohol, metal ions, hormones, heat, cold, and the like. For example, isopropylthio-β-galactoside (IPTG) and lactose are inducers of the lac UV5 promoter, while Larabinose is a suitable inducer of the arabinose (araB) promoter.

As used herein, the term "inducible promoter" refers to a promoter that, upon binding to a transcription factor, directs transcription at an increased or decreased rate.

As used herein, the term "controlled" refers to a nucleic acid sequence operably linked to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence. This requires that the attached DNA sequences are adjacent to each other and that the secretory peptides are in the reading frame.

Accordingly, E. coli is transformed using pBR322, a plasmid derived from E. coli species. See eg, Bolivar et al., Gene, 2:95 (1977). Plasmid pBR322 contains genes conferring resistance to ampicillin and tetracycline to allow identification of transformed cells. Plasmid pBR322 or another microbial plasmid or phage also typically contains, or is modified to contain, promoters that can be used by the bacterial organism to express selectable marker genes. The expression system of the invention comprises a nucleic acid encoding a recombinant protein encoding victronectin operably linked to a first inducible promoter.

The expression system of the present invention also comprises a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter.

As used herein, the term "secretory signal peptide" or "signal peptide" refers to a peptide that can be used to secrete a recombinant polypeptide into the periplasm of a bacterial host. The signal for the heterologous polypeptide may be endogenous to the bacteria, or it may be exogenous, including signals specific to the polypeptide produced by the host bacteria. Typically, the signal sequence can be a component of the vector or part of the polypeptide DNA inserted into the vector.

Preferably, the vector has a nucleic acid encoding a signal sequence at the N-terminus of the T4 lysozyme protein. Preferably, the secretory signal peptide is pectate lyase B (PelB). In one embodiment, the PelB sequence may be from Erwinia spp. Accordingly, disclosed herein is a gpe with a pelb signal sequence that comprises the sequence of SEQ ID NO. 2. The secretion signal sequence can be directly linked to the nucleotide sequence encoding the recombinant polypeptide, e.g. vitronectin. Alternatively, between the secretory signal peptide and the nucleotide sequence encoding the recombinant polypeptide, e.g. vitronectin, may be a spacer. The spacer may comprise more or less than 9 nucleotides, for example 5 to 20 nucleotides.

For the construction of vectors containing one or more of the components described above, well-known ligation techniques are used. The DNA fragments are broken down and religated into the form needed to create suitable vectors.

Confirmation that the constructed plasmids and vectors contain the correct sequences can be obtained by transforming E. coli K12 strain 294 (ATCC 31 446) or other suitable strains and selecting the transformants for resistance to ampicillin or tetracycline.

In one embodiment, the vector expresses vitronectin under the control of the IPTG-inducible pT7 promoter or the IPTG-inducible lacUV5 promoter. Preferably, the vector is a pET29 vector. In one embodiment, the vector is a pBR322 vector. In one embodiment, the vector is pET29 - optVTN as described herein.

In one embodiment, the vector expresses a T4 phage lysozyme containing an N-terminal PelB signal sequence under the control of a T7 polymerase promoter. In one embodiment, the vector is pLATE11 (cat. K1241, Thermo Fisher). In one embodiment, the vector is pACYC184-PT7-pelB-gpe-lacI as described herein. In one embodiment, the T7 polymerase promoter contains two flanking lac operator sequences for tight control of gene expression and may further contain one or more of (i) a Ptet promoter to downregulate basal expression from the T7 promoter, (ii) a T7 terminator that terminates transcription from the T7 promoter and (iii) the lac repressor, which ensures tight control of basal expression from the T7 promoter. In one embodiment, the T7 promoter can be subcloned into the pACYC184 vector, which contains the p15A origin of replication and is compatible with plasmids containing the ColEI ori.

In one embodiment, the vector expresses a T4 phage lysozyme containing an N-terminal PelB signal sequence under the control of the araB promoter. In one embodiment, the vector is a pBR322 vector. In one embodiment, the vector is a pET29 vector. In one embodiment, the vector is pLysS-lacl-PcsP-pelBgpe, where lacl-PcsP is the cold-inducible promoter csp together with the IPTG-inducible operon lacl. In one embodiment, the vector is pACYC184lacl-Pcsp-PelB-gpe.

The host cell

The method of the invention uses a host cell, i.e. i.e. E. coli transformed or transfected with the expression systems described above.

As used herein, the term "transformed" or "transformation" refers to the introduction of DNA into an organism such that the DNA is replicated either as an extrachromosomal element or as a chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells.

Recombinant cells for use in the methods of the present invention can be produced using recombinant DNA techniques known to those skilled in the art (see, e.g., Kotewicz, M.L., et al., Nucl. Acids Res. 16:265 (1988); Soltis , D. A., and A. M., Proc. Acad. Sci. 85:3372-3376). Such recombinant cells can be cultured by standard microbiological techniques using culture medium and incubation conditions suitable for the growth of certain types of active cultures, which are well known to those skilled in the art (see, e.g., Brock, T.D., and Freeze, H., J .Bacteriol. 98(1):289-297; Imahori, K. 24(1):102-112.

Transformation refers to the introduction of DNA into an organism such that the DNA is replicated as an extrachromosomal element or as a chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), is commonly used for bacterial cells that contain essential cell wall barriers. Another transformation method uses polyethylene glycol/DMSO as described in Chung and Miller, Nucleic Acids Res., 16: 3580 (1988). Another method is the use of a technology called electroporation.

As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all include progeny. Thus, the words "transformants" and "transformed cells" include the primary cell and the cultures derived therefrom, regardless of the number of passages.

Here, Escherichia coli or E. coli' means a gram-negative, facultative anaerobic rod-shaped coliform bacterium of the genus Escherichia. E. coli host strains for fermentation of recombinant DNA products are well known in the art and include E. coli B (eg, BL21(DE3) or derivatives thereof, E. coli W3110 (ATCC 27,325), E. coli 294 ( ATCC 31,446) and E. coli X1776 (ATCC 31,537) E. coli B is the preferred host for the methods of the invention.

According to the method of the invention, E. coli can be transformed with one or two expression vectors containing a nucleic acid encoding a recombinant protein and a nucleic acid encoding a T4 lysozyme protein. In one embodiment, bacterial cells are transformed with two vectors, respectively, containing a nucleic acid encoding a recombinant protein and a nucleic acid encoding a T4 lysozyme protein. In an alternative embodiment, the nucleic acid encoding the recombinant protein and the nucleic acid encoding the T4 lysozyme protein are present in a single vector used to transform bacterial cells.

E. coli cells are transformed with the expression vector(s) of the present invention described above and cultured in conventional nutrient media modified to be suitable for inducing the various promoters, if induction is to be effected.

In one embodiment, the host cell is E. coli, preferably E. coli B strain, transformed with a vector expressing vitronectin under the control of the IPTG-inducible PT7 or IPTG-inducible lacUV5 promoter. The vector can be any suitable vector such as pET29 or pBR322 vector.

In one embodiment, the host cell is E. coli, preferably an E. coli B strain such as BL21(DE3), transformed with a vector expressing a T4 phage lysozyme containing an N-terminal PelB signal sequence under the control of the PT7 or araB promoter. The vector can be any suitable vector, such as the pBR322 vector.

In one embodiment, the host cell is E. coli, preferably E. coli B strain, transformed with a vector expressing vitronectin under the control of an IPTG-inducible promoter, such as the PT7 or lacUV5 promoter, and T4 phage lysozyme containing the N-terminal PelB signal sequence under the control promoter, such as the pT7 promoter or the araB promoter. The vector can be any suitable vector described herein, such as the pBR322 vector.

Cell culture

The transformed host cells described above are fermented to express a nucleic acid encoding a recombinant protein (eg, vitronectin) and a nucleic acid encoding T4 lysozyme.

As used herein, the terms "fermentation" and "cultivation" are used interchangeably to refer to cells grown in large quantities in a culture medium. These include an exponential phase characterized by cell doubling, massive growth, and a stationary phase where the rate of cell growth and death rate are equal, often due to a growth-limiting factor such as depletion of a key nutrient and/or an inhibitory product such as an organic acid. formation.

In the process of the invention, the expression of each of the recombinant protein and T4 lysozyme is controlled by induction of associated promoters. Induction of each protein (eg, vitronectin) and T4 lysozyme promoters can be performed sequentially or simultaneously.

Induction of the expression of the nucleic acid encoding the recombinant protein and T4 lysozyme is carried out, preferably by adding an inducer to the culture. Usually, the inducer is added after the cells are cultured until a certain optical density is reached, e.g. OD600 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or greater. In one embodiment, the T4 lysozyme inducer is added together with the recombinant protein inducer. In one embodiment, the T4 lysozyme inducer is added sequentially after induction of the recombinant protein.

In one embodiment, induction of each recombinant protein and T4 lysozyme promoters occurs simultaneously. In this embodiment, the promoters of T4 lysozyme and the nucleic acid encoding the polymerase may be identical. Although the promoters can be any promoter suitable for this purpose, it is preferred that the promoters for the recombinant protein and the polymerase are IPTG-inducible promoters, such as the T7 promoter.

In one embodiment, the induction of each of the recombinant protein and T4 lysozyme promoters is sequential, with induction of the T4 lysozyme promoter beginning about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours after induction of the recombinant protein promoter. In this embodiment, the promoters of the T4 lysozyme-encoding nucleic acid and the polymerase-encoding nucleic acid must be different so that expression of the nucleic acid-encoded recombinant protein is induced before expression of the nucleic acid-encoded T4 lysozyme or at a much higher level when the promoters are induced. Although the promoters can be any promoter suitable for this purpose, it is preferred that the promoters for the recombinant protein and the polymerase are an arabinose promoter and an IPTG-inducible promoter, respectively.

When the T4 lysozyme inducer is added sequentially, the inducer is typically added after the desired amount of recombinant protein has accumulated (eg, as determined by optical density reaching a target amount that has been observed in the past and that correlates with the accumulation of the desired polypeptide). Typically, induction of T4 lysozyme occurs after inoculation after about 75-90%, preferably about 80-90%, of the total fermentation time. For example, induction of the T4 lysozyme promoter can occur from about 30 hours, preferably 32 hours, to about 36 hours after inoculation in a 40 hour fermentation process. For example, induction of the T4 lysozyme promoter can occur about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours after induction of expression of the recombinant protein (eg, vitronectin).

Induction of T4 lysozyme permeabilizes the cell to form a spheroplast that secretes a recombinant protein (eg, vitronectin). As used herein, the term "permeabilizes" refers to making openings in the cell wall without removing it completely. A "spheroplast" is a cell in which the cell wall of a gram-negative bacterium is partially lost and becomes spherical. Spheroplastization can be performed by treating gram-negative bacteria with lysozyme, penicillin, or the like. Since the cell membrane of spheroplastized Gram-negative bacteria is partially lost, information about the interaction between the target protein and the polypeptide (electrophysiological changes) can be analyzed by electrophysiological method. Moreover, the same polypeptide is present in high concentration in the spheroplast of a single cell, which is a favorable condition for measuring the activity of the polypeptide on the target protein. Generally, spheroplasts that retain substantially all of the nucleic acids present in the spheroplast while allowing intracellular proteins (including recombinant protein) to move across the spheroplast membrane.

Generally, the culturing step is carried out at a high cell density, that is, when the cell density is between 15 and 150 g dry weight/liter, preferably at least about 40, more preferably about 40-150, most preferably about 40 to 100. In addition, culturing can be using any, even very large volumes of 100,000 liters. The volume is preferably about 100 liters or more, preferably at least about 500 liters, and most preferably from about 500 liters to 100,000 liters.

The E. coli cells used to produce the polypeptide of interest described in the present invention are cultured in a suitable medium in which the promoters can be induced as conventionally described, e.g., Sambrook et al.

The terms medium, cell culture medium, and fermentation medium are used interchangeably herein and refer to a solution containing nutrients required by bacteria, i.e. i.e. E. coli, grow. Generally, the cell culture medium contains essential and essential amino acids, vitamins, energy sources, lipids and micronutrients that the cell needs for minimal growth and/or survival. Preferably, the medium is chemically defined in that all its components and their concentrations are known. If cells are cultured in a fed-batch mode as described below, the term "culture medium" refers to both basal and nutrient medium, unless otherwise specified. The pH value of the medium can be anywhere between pH 5 and 9, depending mainly on the host organism.

In order to accumulate the recombinant gene product, i.e. i.e. recombinant protein such as vitronectin, the host cell is cultured under conditions sufficient to accumulate the gene product. Such conditions include, for example, temperature, nutrients, and cell density conditions that allow protein expression and accumulation within the cell. Moreover, such conditions are those under which the cell can perform the basic cellular functions of transcription, translation, and the movement of proteins from one cell compartment to another for secreted proteins, as is known to those skilled in the art.

Different cell culture strategies are available, including closed vessel culture, perfusion culture, continuous culture, and fed-batch culture.

In one embodiment of the methods of the present invention, the temperature is changed during the cultivation process from a first temperature to a second temperature, i.e. i.e. the temperature is actively reduced. Preferably, the second temperature should be lower than the first. The first temperature can be 37 °C ± 0.2 °C. The second temperature may be in the range of 25°C to 36°C, preferably in the range of 28°C to 36°C, more preferably in the range of 28°C to 32°C, most preferably in the range of from 29 °C to 31 °C. The second temperature can be 29 °C, 30 °C or 31 °C. In one embodiment, the first temperature is 37°C and the second temperature is 30°C.

Gene expression can be measured in a sample directly, for example, using a conventional Northern blot technique to quantify mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)).

In some embodiments, the bacteria are cultured in a bioreactor. As described herein, the methods of the invention have been shown to be particularly suitable for large-scale production, such as in a bioreactor or in vessels larger than 1 L. In some embodiments, the bioreactor is at least about 1 L volume, at least about 5 L volume, at least about 10 L volume, at least about 15 L volume, at least about 20 L volume, at least about 30 L volume, at least about 40 L volume, at least about 50 l volume, at least about 100 l volume, at least about 200 l volume, at least about 250 l volume, at least about 500 l volume, at least about 750 l volume, at least about 1000 l volume, at least about 1500 l volume, at least about 2000 l volume, at least about 2,500 l volume, at least about 3,000 l volume, at least about 3,500 l volume, at least about 4,000 l volume, at least about 5,000 l volume, at least about 7,500 l volume, at least about 10,000 l volume, at least 15,000 l volume , at least 20,000 l volume, at least 30,000 l volume, at least 50,000 l volume, at least about 100,000 l volume, at least about 150,000 l volume, at least about 200,000 l volume, at least about 250,000 l volume, at least about 300 000 l volume, at least about 350,000 l volume, at least about 400,000 l volume, at least about 450,000 l volume or at least about 500,000 l volume.

Since the recombinant polypeptide (eg, vitronectin) is successfully secreted into the cell culture supernatant during the culture process, the recombinant polypeptide can be isolated at the end of the culture process by separating the cell culture supernatant comprising the recombinant protein. This method successfully eliminates the need to lyse the transformed cells in order to isolate the recombinant protein. This reduces the amount of cellular debris and the release of nucleic acids and their molecules, which can affect the quality of the recombinant protein product. Collecting the cells marks the end of the fermentation/cultivation. Cells can be harvested at any point in the fermentation considered sufficient to complete the fermentation process and yield the expressed recombinant protein. Cell harvesting can occur 10 to 60 hours after induction of conditions allowing expression of the recombinant protein. For example, cells can be harvested 15 to 40 hours after induction. At the time of cell harvesting, the fermented culture medium includes cells that have been autolyzed and membrane permeabilized. For example, about 50% or more of the harvested cells may have been autolyzed. Alternatively, about 50%, 55%, 60%, 65%, 70%, 80% or 85% of the harvested cells were autolysed.

As used herein, the terms "cell lysis," "lysis," or "lysing" refer to the release of DNA from a cell by complete disruption of the cell membrane. If a cell loses its DNA, it can be considered non-viable, i.e. dead. i.e. dead In contrast, "membrane permeability" and "permeability" refer to disruption of the integrity of the cell membrane that results from leakage of the recombinant product into the cell culture supernatant without complete lysis of the cell membrane. The method of the invention makes it possible to isolate the recombinant protein without completely disrupting the cells. More precisely, the method of the invention provides for the isolation of the recombinant protein, which must be carried out without mechanical disruption of the cell; without adding an exogenous enzyme that degrades the cell wall; without transforming the cell with expression vectors containing a nucleic acid encoding an exogenous enzyme that disrupts the cytoplasmic membrane, e.g. T4 choline protein. Accordingly, in embodiments, the method of the invention does not include any of the following steps: (i) mechanical cell disruption; (ii) addition of an exogenous enzyme that degrades the cell wall; and (iii) transforming the cell with expression vectors containing a nucleic acid encoding an exogenous enzyme that disrupts the cytoplasmic membrane, e.g. T4 choline protein.

Methods are known to monitor both release of the recombinant product and cell lysis. Common to both is the detection of marker molecules, such as a cellular product that indicates shedding or DNA lysis. Methods for measuring release include photometry (Dietzsch, J. Biotechnol., 163, pp. 362-370 (2013); Wurm, Eng. Life Sci., 17 pp. 215-222 (2017)) and high performance liquid chromatography (Amand, Biotechnol., 30, pp. 249-255; Kaiser Eng., 8 pp. 138 (2008); Methods for measuring lysis include colorimetric assays with hand-held photometers (at-line) or coupled with liquid handling systems (at/online) (Rajamanickam, Anal. Bioanal. Chem., 409, pp. 667-671 (2017)) and infrared /raman spectroscopy (Abu-Absi, Pharm. Bioprocess., 2 , pp. 267-284 (2014); Buckley, Appl. Spectrosc., 71 , pp. 1085-1116 (2017); C^en, Anal. Bioanal. Chem., 409 pp. 651-666 (2017); Biotechnol., 33, pp. 285-298 (2017). Autolysis can be measured indirectly by DNA concentration in purified harvested cells or by capacity.

Secreted recombinant proteins are sensitive to cell culture instability, such as aggregating and forming improper spatial structure after secretion/cell lysis. In certain embodiments, a stabilizing agent is added to the cell culture. The stabilizer can be added before, during or after fermentation. Examples of suitable stabilizers include potassium chloride, DTT, sodium chloride, trehalose, sucrose, glycine betaine, mannitol, potassium citrate, CuCl2, proline, xylitol, NDSB 201, CTAB, K2PO4 Na2SO4, and Brij 35. In certain embodiments, the stabilizing additive may include amino acids , such as arginine.

In certain embodiments, a nucleic acid inactivating agent (eg, benzonase) can be used to remove free nucleic acid (DNA and/or RNA) from the cell culture. The nucleic acid inactivating agent can be added before, during or after fermentation. Preferably, the nucleic acid inactivating agent is not centrifuged prior to clarifying the cell culture. Thus, any method of the invention may include (i) adding a nucleic acid (eg, DNA) inactivating agent (such as benzonase) to the cell culture in the absence of a further centrifugation step.

Clarification

According to the methods of the present invention, autolyzed and/or permeabilized cells are separated from the culture medium prior to isolation and purification of the product from the cell medium by clarification.

As used herein, the terms "clarify", "clarification", "clarification step" refer to a process comprising one or more steps used to remove solid impurities from a cell culture to obtain a liquid crude filtrate containing a recombinant protein.

The efficiency of the clarification step is critical to facilitate subsequent processing steps that require the purification of the biomolecule of interest. Parameters that can affect the clarification step include total cell concentration, cell viability, initial turbidity of the cell culture for clarification, concentration of biomolecule produced by cultured cells, and size of cell debris. As used herein, the term total cell concentration (TCC) refers to the number of cells in a given culture volume. As used herein, the term viable cell concentration (VCC) refers to the number of viable cells in a given volume of culture as determined by standard known viability assays. As used herein, the term "viability" refers to the percentage of living cells. Generally, the higher the TCC, the greater the biomass to be removed from the cell culture and the greater the effect on clarification. As used herein, the term "turbidity" refers to the turbidity or turbidity of a liquid caused by a large number of individual particles. In particular, turbidity indicates the amount of matter and small particles in a liquid that can scatter light. Typically, cell culture turbidity correlates with cells in culture, cell debris, nucleic acids, and host cell protein (HCP) in addition to the recombinant polypeptide.

The clarification step reduces the initial turbidity of the fermented cell culture to a lower turbidity of the clarified cell culture to produce a clarified cell culture with the highest concentration of the biomolecule of interest and a lower amount of other cell culture material.

In one embodiment, the initial turbidity of the fermented cell culture is greater than about 10,000 NTU, about 20,000 NTU, about 30,000 NTU. In some embodiments, the initial turbidity of the fermented cell culture is between about 10,000 NTU and about 150,000 NTU, and more specifically includes between about 30,000 NTU and about 100,000 NTU.

In one embodiment, the clarified cell culture has a turbidity equal to or less than about 40, 30, 25, or 20 NTU. The clarified cell culture preferably has a turbidity of about 20 NTU or less.

For the clarification method to be effective, it is also important to maximize throughput. As used herein, the terms "capacity" or "carrying capacity" or "capacity" may be used interchangeably and refer to the volume clarified in a functional unit of clarification, such as the volume filtered through a filter, more specifically the volume normalized by the filter area (L/m ² ) .

Clarification of the cell culture of the invention includes a primary clarification step. As used herein, the terms "primary clarification" refer to the removal of large particles such as whole cells and cell debris. In embodiments, the primary clarification may be followed by a secondary clarification step. As used herein, the term "secondary clarification" generally refers to the removal of smaller particles, such as particles smaller than whole cells. Each of the primary and secondary clarification stages uses a filter as the functional unit of clarification.

In a preferred embodiment, the primary clarification step and/or the secondary clarification step is microfiltration. Typically, microfiltration is used to separate particles of 0.1-10 μm in size from the solution. It is most often used to separate a polymer with a molecular weight of 1 χ 10 ⁵ g/mol. In addition, microfiltration can be used for sediments, protozoa, large bacteria, etc. remove In the present invention, microfiltration can easily be used to remove whole cells and cell debris. Typically, the microfiltration process is carried out using a pressure pump or a vacuum pump with a speed of 0.1-5 m/s, preferably 1-3 m/s, and a pressure of 50-600 kPa, preferably 100-400 kPa.

In a preferred embodiment, the primary clarification step and/or the secondary clarification step is depth filtration. As used herein, the term "depth filtration" refers to a technology that utilizes filters of a certain porosity to trap media particles throughout the filter and not just on the surface of the filter. Depth filters are typically composed of cellulose fibers or synthetic polymer fibers such as polyacrylic or polystyrene. These fibers form a three-dimensional network with a certain porosity. A depth filter may be suitable for primary and/or secondary extraction, or only for primary or secondary extraction. Filters suitable for primary and/or secondary regeneration are also known as single-stage filters. Single-stage filters can be used to perform both primary and secondary extraction, or they can be used as filters for primary extraction and in combination with the subsequent use of a secondary extraction filter. In certain embodiments, this depth filter is positively charged. The interaction between the positively charged filter and the colloidal particles in the fermented cell culture successfully allows the removal of such small impurities, while the larger particles are retained by the porosity of the filter matrix.

The filter may be composed of a variety of materials well known in the art. Such materials may include, but are not limited to, polymeric materials such as nylon, cellulosic materials (eg, cellulose acetate), polyvinylidene fluoride (PVDF), polyethersulfone, polyamide, polysulfone, polypropylene, and polyethylene terephthalate. In certain embodiments, filters used to clarify cell lysates may include, but are not limited to, StaxTM depth filter systems (Pall Life Sciences), ULTIPLEAT PROFILE™ filters (Pall Corporation, Port Washington, NY), SUPOR™ membrane filters (Pall Corporation, Port Washington, NY). Commercial depth filters include StaxTM depth filter systems (Pall Life Sciences), Millistak+ Pod depth filter system, XOHC media (Millipore Corporation), Zeta Plus™ depth filter (3M Purification Inc.), and others.

In embodiments, pre-clarification is performed with a first filter having a pore size of at least about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 , 17.0, 18.0, 19.0, 20.0 μm (micrometer) or greater retention limits. In another embodiment, the first filter has a pore size that provides retention limits from about 0.4 μm to about 20.0 μm. In another embodiment, the first filter has a pore size that provides retention limits from about 0.4 μm to about 9.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 0.4 μm and about 3.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 0.4 μm and about 0.8 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 1.0 μm and about 3.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 1.0 μm and about 20.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 1.0 μm and about 9.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 4.0 μm and about 20.0 μm. In another embodiment, the first filter has a pore size that provides retention limits between about 4.0 μm and about 9.0 μm. In a certain embodiment, the first clarification step is performed using a first depth filtration.

The clarification efficiency can be improved by increasing the filter bandwidth. As used herein, the terms "permeability" or "carrying capacity" or "capacity" are used interchangeably and refer to the volume clarified by a functional unit of clarification, such as the volume filtered through a filter, or rather, the volume normalized by the filter area (L/m ² ).

In one embodiment, the maximum throughput of the pre-clarification step is at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 , 850, 900, 950, 1000 L/m2.

In embodiments, the secondary clarification is performed by a second filter, e.g. depth filter with a pore size of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3, 0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0 μΜ or higher retention limits. In another embodiment, the second filter has a pore size that provides retention limits of about 0.4 μm to about 3.0 μm. In another embodiment, the second filter has a pore size that provides retention limits between about 0.4 μm and about 0.8 μm. In another embodiment, the second filter has a pore size that provides retention limits between about 1.0 μm and about 3.0 μm. In a certain embodiment, the second clarification step is performed using a second depth filtering.

In one embodiment, the maximum throughput of the secondary clarification step is at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 L/m2.

In one embodiment of the present invention, a step of pre-treatment of the cell culture is performed prior to primary clarification to increase clarification efficiency, such as flocculation.

As used herein, the term "flocculation" refers to the aggregation, precipitation, and/or agglomeration of insoluble particles such as cellular material, including cells, cellular debris, host cell proteins, DNA, and other components, caused by the addition of a suitable flocculating agent to a fermented cell culture. Increasing the particle size of the insoluble components present in the fermented cell culture improves the efficiency of separation, such as clarification by filtration. Flocculation can be initiated by known methods, including lowering the pH value of the cell culture or adding a flocculating agent. Non-limiting examples of a flocculating agent include calcium phosphate, caprylic acid, divalent cations, or positively charged polymers such as polyamine, polyethyleneimine (PEI), chitosan, or polydiallyldimethylammonium chloride (e.g., pDADMAC) that cause particle aggregation through interactions with the negatively charged cell surface and cell remains. In one embodiment, the flocculating agent is a positively charged polymer, preferably polyethyleneimine (PEI). In embodiments, the flocculating agent is PEI and is added to the fermented cell culture at about 0.10% (v/v) to about 0.50% (v/v). In embodiments, the flocculating agent is PEI and added to the fermented cell culture at a concentration of from about 0.15% (v/v) to about 0.25% (v/v). In embodiments, the flocculating agent is added to the fermented cell culture at a concentration of about 0.10% (v/v), 0.15% (v/v), 0.20% (v/v), 0.25% (v/v ) or 0.30% (v/v). In embodiments, the flocculating agent is PEI and added to the fermented cell culture at a concentration equal to about 0.15% (v/v) or about 0.25% (v/v).

In one embodiment, the flocculating agent is added directly to the fermented cell culture liquid at the end of the fermentation, and the flocculated material, including the cells, is removed from the cell culture liquid by filtration. In one embodiment, the flocculating agent is added prior to harvesting the cells.

In preferred embodiments, fermentation and clarification are performed as a closed system method. As used herein, the term "closed system method" refers to a method or process that is performed in closed sterile systems under controlled environmental conditions without any risk of external contamination from the operator or the laboratory environment. It is especially important to use a closed system when creating products of the GGP class. In substantially preferred embodiments, the methods of the invention are closed system methods that do not involve centrifugation, since centrifugation cannot be performed in a closed system and requires transfer of an intermediate product (lysed cells) to centrifugation vessels. Post-fermentation clarification can be carried out by transferring the fermented culture from the bioreactor in a flow in closed tube systems to the clarification filters, without any operator contact.

Further processing of the purified protein

In embodiments, the recombinant protein can be isolated directly from the clarified culture medium. Isolation of the recombinant protein may be followed by purification to ensure adequate purity of the recombinant protein. Various methods of protein purification are well known to those skilled in the art.

Suitable purification techniques include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, gel electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, gel filtration, liquid chromatography (LC ), high performance LC (HPLC), high performance LC (FPLC), hydroxyapatite chromatography, lectin chromatography and immobilized metal affinity chromatography (IMAC). Most preferably, recombinant proteins are purified by liquid chromatography techniques including ion exchange, affinity and gel filtration.

Thus, the methods of the invention provide substantially pure recombinant proteins. Substantially pure, as used herein, refers to a preparation or sample that is substantially free of impurity components, proteins, etc.

Throughout the description and statements of this specification, the words "comprising" and "comprising" and their variations mean "including but not limited to" and do not refer to (and do not exclude) other parts, accessories, components or steps. Throughout the description and definition of this specification, the singular includes the plural unless the context otherwise requires. In particular, when the indefinite article is used, the specification shall be understood to include both the plural and the singular unless the context otherwise requires.

Signs, integers, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the invention are to be understood as applicable to any other aspect, embodiment, or example described herein unless inconsistent therewith. All features disclosed in this specification (including any accompanying definitions, abstracts and drawings) and/or all steps of the method or process so disclosed may be combined in any combination except combinations in which at least some of such features and/or stages are incompatible with each other. The invention is not limited to the details of any of the above embodiments. The invention includes any new or any new combination of features disclosed in this specification (including any accompanying definitions, abstracts and drawings), or any new or any new combination of steps in any method or process so disclosed.

The reader's attention is directed to all articles and documents filed with or prior to this specification relating to this application and open to the public in connection with this specification, and the contents of all such articles and documents are incorporated herein by reference.

EXAMPLES example

Construction of vitronectin (hereinafter referred to as VTN)

The target vitronectin gene (sequence disclosed herein as SEQ ID NO: 1) was cloned into the pET vector under the inducible promoter PT7 (Figure 1A). NCBI Sequence ID: AAH05046.1, Protein Name: Vitronectin, Target Gene Origin: Homo sapiens.

Construction of the T4 bacteriophage lysozyme gene gpe

The T4 bacteriophage lysozyme gene gpe was cloned into a tightly controlled pLATE11 vector (cat. no. K1241, Thermo Fisher) to reduce lysis gene expression in uninduced cells. In addition, the pelB signal peptide for translocation of the produced protein to the periplasmic space was fused to the N terminus of Gpe. A fragment containing the transcription terminator rrnBT1-T2 prevented basal gene expression from vector-derived promoter-like elements, the T7 RNA polymerase promoter that drives transcription of the cloned gpe gene, and two flanking lac operator sequences provide tight control of gene expression, the Ptet promoter reduces constitutive expression from the T7 promoter, the T7 terminator, which stops transcription from the T7 promoter, and the lac repressor, which ensures tight control of constitutive expression from the T7 promoter, were subcloned into the pACYC184 vector, which contains the p15A origin of replication and is compatible with plasmids containing the ColE1 ori. The recombinant plasmid was named pACYC184-PT7-pelB-gpe-lacI (Figure 1B).

This example used a modified polypeptide called Gpe. The sequence is derived from Eschericia coli T4 bacteriophage lysozyme and is disclosed herein as SEQ ID NO. 2 - NCBI reference sequence: NP_049736.1, protein name: glycoside hydrolase family protein (Escherichia phage T4). PelB signal sequence: 1 - 22, T4 bacteriophage lysozyme 23 - 164.

Expression strain preparation

One Shot™ BL21 (DE3) E. coli cells were transformed with plasmids pET29-optVTN and pACYC184-PT7-pelB-gpe-lacI and plated on LB non-animal origin (AOF) agar with kanamycin (50 g/L) and 25 μg/ml chloramphenicol.

The transformation was based on the temperature shock method. A research cell bank (RCB) was produced by culturing transformants in liquid TB4 AOF medium with the required antibiotics at 37 °C and 220 rpm. A sterile 50% glycerol solution was used to produce 25% glycerol RCB, which was aliquoted into cryogenic tubes and stored at −70°C.

Growing medium

Propagation of transformants and plasmids was performed in solid and liquid LB animal-free (AOF) medium containing plant peptone (10 g/l), yeast extract (5 g/l), NaCl (10 g/l), and solid medium 15 g/l of microbiological agar, as well as the necessary antibiotics. Batch-feed and closed-cell cultivation was carried out in glycerol-based AOF broth (TB4 AOF and TB AOF) containing (per liter): vegetable peptone 12 g, yeast extract 24 g, (NH4)2SO4 2.68 g, KH2PO4 2, 3 g, K2HPO4 12.5 g and glycerol from 4 to 10 g. In addition, before cultivation, the TB medium was supplemented with the following sterile solutions: 2 ml (1 M) MgSO4 and 2 ml of a micronutrient solution with the composition (per liter): CaCl2 χ 2H2O 0.5 g, ZnSO4 χ 7H2O 0.18 g, MnSO4 χ H2O 0.1 g, Na2-EDTA 20.1 g, FeCfe x 6H2O 16.7 g, CuSO4 x 5H2O 0.16 g, CoCl2 x 6H2O 0.18 g; as well as 100 μl L-1 thiamine hydrochloride (1 M), 1 ml 5-kanamycin (50 mg ml/l) and 1 ml chloramphenicol (30 mg/l).

Expression and autolysis conditions in shake flasks

To select an optimal E. coli strain for VTN expression, E. coli JS007 and Invitrogen One Shot™ BL21 (DE3) chemically competent E. coli cells were transformed with both targeting recombinant plasmids pET29-optVTN and pACYC184-PT7-pelB-gpe-lacI. The level of protein expression was similar in both strains (Fig. 3B), but BL21(DE3) cells showed higher biomass yield (Fig. 3A), so the BL21(DE3) strain was selected for further use in cell banking. Cells and medium fractions were separated by centrifugation at 30 m in., 14000 rpm, at 4 °C. Medium fractions were stored at 4 °C. Cell samples taken from the culture flask were resuspended in lysis buffer at the following ratio: 1 g of biomass was resuspended in 10 ml of lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA, 0.1 mg/ml lysozyme). The biomass was sonicated for 60 seconds at 4 °C. Soluble and insoluble protein fractions were separated by centrifugation for 20 min, 14000 rpm, 4 °C. The total protein fraction is a suspension of cell debris (crude extract). Cell samples for SDS-PAGE separation were prepared as follows: 20 μl of crude extract sample, 25 μl of 4x SDS-PAGE loading buffer, 5 μΙ of 2M DTT, and 50 μΙ of deionized water for a final sample volume of 100 μΙ. Samples of SDSPAGE separation medium were prepared as follows: 70 μΙ growth medium, 25 μΙ 4x SDS-PAGE loading buffer, 5 μΙ 2 M for a final sample volume of 100 μΙ. The samples are heated for 10 minutes at 95 °C. 10 μΙ of sample was applied to each geHo lane.

Expression and autolysis conditions in the bioreactor

Cultivation medium and nutrient solution - Propagation of the preinoculate and inoculum was carried out in a rotation created on a gerose-based medium without animal feed (AOF) (TB4 AOF) with the composition (Htre): vegetable peptone 12 g, barley extract 24 g, (NH4)2SO4 2 ,68 g, KH2PO4 2.3 g, K2HPO4 12.5 g and augaHnis g^eroMs 4 g. In addition, before seeding, the TB4 medium was treated with the following sterile solutions: 3 ηηΙ/Ι (1 M) MgSO4 and 3 m^ microelement solutions (TES) with the composition (Htre): Cad2 x 2H2O 0.5 g, ZnSO4 x 7H2O 0, 18 g, MnSO4 x H2O 0.1 g, Na2-EDTA 20.1 g, FeCfe x 6H2O 16.7 g, CUSO4 x 5H2O 0.16 g, Cod2 x 6H2O 0.18 g; as well as 3 m^ of thiamine hydrocNoride (10 g/Ι), 1 m^ of kanamycin (50 mg ml·^ and 1 m^ of cNoramfenikoho (25 mg/Ι). Spin medium with 15 g of gHceroHo (TB15 AOF).

PreinokuHato preparation

Single cotton from plates of E. coli BL21(DE3) [pET29-optVTN and pACYC184 PT7-PeBgpe-tacI] transformants were transferred to 250 ml ta^a shake flasks with 50 ml TB4 AOF medium with papHds and antibiotics and incubated for 8 ± 1 h · at 37 °C / 230 rpm. The optical density (OD600) of the maize was measured at the end of cultivation, and the seeding volume for seeding the cob was calculated using the formula: 4/OD preinokuHatas = mk

Preparation of InokuHato

The calculated preincubate volume was transferred to 2000 ml shake flasks with 500 ml of TB4 AOF medium with papHds and antibiotics and incubated for 17 ± 1 vat at 25 °C / 230 rpm. The optical density (OD600) of the corn is measured at the end of cultivation. The seeding volume for bioreactors is 2% of the total medium volume.

Bioreactor processes

The batch stage was performed at 300 L S.U. in a fermenter (250 l medium). The growth dynamics of the culture was monitored by optical density measurements at a wavelength of 600 nm. Samples were taken from the bioreactors at various stages of the process and analyzed. Bioreactors were seeded with an overnight inoculum (2% by volume). Initial culture parameters prior to induction of target protein and concomitant lysis were as follows: dissolved oxygen (DO) control was maintained at 30% by automatic cascade: air flow (75 to 250 L/min); mixing speed (from 100 to 375 rpm); pure oxygen flow (from 0 to 160 l/min.). The culture pH was adjusted at 7.0 ± 0.05 by automatic addition of NH4OH (25%) or H3PO4 (42.5%). The culture temperature was maintained at 37 °C before VTN induction (VTN induction point: 3.5-5.5 OD). Before induction in 0.1 mM IPTG, the temperature was lowered from 37 to 25 °C in all processes. Addition of IPTG simultaneously induces expression of target proteins and expression of lysozyme in cells. 3% OD was determined 0.5 h after induction. 2-4 hours after induction, 18750000 u of benzonase (universal nuclease) is added, then the mixer is changed to at least 100 rpm, minimum air flow to 40 l/min. Fermentation continues at 17 - 22 hours. after induction. At the end of the fermentation process, 18750000 u of benzonase (universal nuclease) is added and stirred for 15 min.

Autolysis and expression analysis of VTN protein

Cells and media fractions were separated by centrifugation for 30 min, 14000 rpm, 4 °C. Medium fractions were stored at 4 °C. Cell samples taken from the culture flask or fermenter were analyzed by SDS-PAGE electrophoresis. Cells were sampled at different culture times (pre-induction and post-induction). Soluble and insoluble protein fractions were separated by centrifugation for 20 min, 14000 rpm, 4 °C. The soluble protein fraction or supernatant after centrifugation showed the efficiency of the autolysis process and how much of the protein was soluble. The insoluble fraction is unbroken cells and insoluble proteins. Cell samples for SDS-PAGE separation were prepared as follows: 20 μl of crude extract sample, 25 μl of 4x SDS-PAGE loading buffer, 5 μl of 2M DTT, and 50 μl of deionized water so that the final sample volume was 100 μl. Media samples for SDS-PAGE separation were prepared as follows: 70 µl culture medium, 25 µl 4 χ SDS-PAGE loading buffer, 5 µl 2 M DTT so that the final sample volume was 100 µl. The samples were heated for 10 minutes at 95 °C. 10 µl of sample was loaded into each 4-12% SDS35 gradient

PAGE gel strip track.

Clarification

For P100 and KS50P media, using a pore size of 1.0 - 3.0 μm, it was found that cells and cell debris of different optical densities are removed. Vitronectin fermentation culture efficiently produces a clear, clarified supernatant. The experiments were repeated with a production-scale (150 l) grown vitronectin fermentation culture and it was calculated that 0.6-0.7 l fermentation culture (OD 600 nm = 15) can be filtered with a 0.05 m ² P100 and KS50P media filter. The results of the experimental series development were extrapolated and the experiment was carried out with a 150 L fermentation culture (OD 600 nm = 17) and 7 x 2 m2 StaxTM 1.0 - 3.0 μm, P100 medium, depth filter capsules. Given that no membrane fouling was observed and a clear filtered supernatant was obtained, these conditions could be maintained on a production scale.

To remove the remaining cell debris and prepare the supernatant for subsequent steps, the clarified cell culture was filtered through a 0.2 μm filter. A 1 m2 membrane area Supor® EX class ECV filter was tested and all 150 L of clarified solution was successfully filtered.

For further use, the buffer solution in the clarified medium was replaced by ultrafiltration. Ultrafiltration was performed in a tangential flow filtration system (TFF) using a TFF filter with a pore size of 30 kDa. The filtration area was 0.01 m2, based on 1 L of medium solution.

In detail, the clarification process of E. coli autolysate involved the following steps (Figure 6).

Starting material: 250 L of cell lysed cell culture, after 16-18 hours of target protein expression in stirred tank reactors. Final OD of cells/cell debris ~15-30 OD. Analysis showed that the induced cell lysis resulted in a cell lysis efficiency of 75-80% based on total cell volume.

During all procedures in a stirred bioreactor (HyperformaTM 300 l, Thermofisher Scientific), all automatic controls in the fermentation vessel: air/O2 flow, pH were in “off mode”. Stirring was maintained at 375 rpm and the temperature was set to 23 °C.

Nucleic acid removal and clarification were performed as described below.

NucleorHydrolysis: universal nuclease is added to the bioreactor in such a way that the final concentration is 250 U/ml, and incubated for 30-60 min. at 23 °C.

Filter preparation: The STAX filter capsule is washed with 10 filter volumes of water, not exceeding a back pressure of 0.2 BAR.

The lysed cell suspension was clarified by filtration through depth filtration capsules (1-3 μΜ (K100P) (Pall, ID 7007836) (approximately 0.05 m ² filter area is required to filter 1 L of fermentation medium) and then filtered through a 0.22 μm filter. capsule (Pall, ID NP6UEAVP1 S) not exceeding 1.2 BAR back pressure Filtration alternatives: K050P (Pall, ID 7007786) Retention 0.4-0.8 μΜ.

E. coli is not a perfect host, especially for closed batch fermentation, because it does not normally secrete proteins into the extracellular medium. The cell disruption step becomes limiting and is not cost-effective on an experimental or industrial scale when the goal is to implement a closed and/or continuous process flow. The present invention provides a cell lysis method which is a programmed cell lysis system based on the expression of the cloned T4 bacteriophage gene gpe to produce a lysis protein. The gene gpe encodes lysozyme, which breaks down the peptidoglycan of the E. coli cell wall.

A self-destructive Escherichia coli has been created that produces a foreign target protein. E. coli was co-transformed with two vector plasmids, the target gene expression vector E. coli BL21(DE3) [pET29-optVTN for expression of the VTN protein and the lysis gene expression vector pACYC184-PT7-pelB-gpe-lacI (or pARApelB-pge). The lysis protein was produced after the expression of the target gene, thus simplifying the cell lysis process. As described here, expression of the cloned T4 phage gene gpe was used to disrupt E. coli that produced VTN as a target protein. Expression of the gpe gene did not induce rapid cell lysis, but weakened the cell wall. Translocation of the lysis protein Gpe from the cytoplasm to the periplasm facilitated the enzymatic degradation of the murein layer, which is responsible for maintaining the rigidity of the cell structure. Microscopic observation revealed that cells producing Gpe in the periplasmic space underwent morphological changes from rod-shaped to elliptical spheroplasts after 4 h (Figure 2).

After 4 h of induction with 0.1 mM IPTG in E. coli BL21(DE3) [pET29-optVTN and pACYC184 PT7-PeB-gpe-lacI] VTN protein was released into the culture medium. Maximal production of VTN protein (about 0.5 g/L) in a shake flask was achieved after overnight expression of E. coli BL21(DE3) [pET29-optVTN and pACYC184 PT7-PeB-gpe-lacI] at 25 °C with lysozyme induction 0.1 mM IPTG was performed together with target gene induction (induction point: 3.5 -5.5 OD) - Figure 3.

The optimized protein expression conditions were successfully transferred to a 3 L bioreactor (Biostat) and scaled up to 300 L SUF. After optimization of the fermentation process, the yield of VTN protein was about 0.1 - 0.2 g/l - Figure 4. and Figure 5.

Clarification process - the provided clarification conditions ensured that all cell debris and insoluble proteins were removed from the solution, leaving only the soluble protein fraction with a target protein (TPR) concentration of about 0.1 - 0.2 g per 1 liter of starting material.

Depth filtration-based clarification of cell debris after nuclease treatment resulted in very small particles of membrane segments. This has been successfully demonstrated using unusually large pore filters. The size of an E. coli cell is: 1.0-2.0 micrometers in length and a radius of about 0.5 micrometers. The primary purification methods that are commonly recommended for E. coli bacteria are tangential flow filtration with a pore size of less than 0.2 micrometers or centrifugation, which is the most common method for clearing cell debris.

The newly developed expression system described here includes genetic elements for the production of target proteins and co-production of lysis enzymes in a controlled manner in the periplasmic space of the cell during the desired growth period, if required. The above experimental data showed that the co-expressed phage lysis enzyme does not completely lyse the cells, but rather forms protoplasts that resemble the cells. This suggested that the translocation of lysozyme from the cytoplasm to the periplasm facilitated the enzymatic degradation of the murein layer, which is responsible for maintaining the firmness of the cell structure. Protoplasts release/secrete target proteins into the culture medium without the help of secretory leader peptides in shake flask and microbial bioreactor production. Protoplasts remain viable for at least 4 hours after induction of lysis enzymes, i.e. y, they de novo produce and secrete the protein of interest, resulting in increased amounts of the target protein in the culture medium over time. VTN protein expression in a fermenter (Biostat A or SUF 300 l) was performed in E. coli with BL21(DE3) [pET29-optVTN protein VTN expression and lysis gene expression vector pACYC184-PT7-pelB-gpe-lacI. Thus, cell lysis pathways mediated by expression of a cloned T4 phage lysis gene are demonstrated here to isolate intracellular target proteins that can be applied to other recombinant proteins by replacing VTN.

A one-pot process in a fermentation vessel that produces recombinant proteins by cell autolysis, followed by protein stabilization and nucleic acid precipitation to eliminate at least 5 separate downstream processing steps: cell harvesting, mechanical and/or enzymatic cell lysis, cell debris separation, precipitation of the nucleic acid and subsequent separation by filtration or centrifugation.

SEQUENCES

VTN protein sequence 1-418 (NCBI Sequence ID: AAH05046.1) vtrgdvftmpedeytvydgeeknnatvheqvggpsltsdlqaqskgnpeqtpvlkpeeeapavev gaskpegidsrpetlhpgrpqppaeeelcsgkpfdaftdlkngslfafrgqycyeldekavrgyp klirdvwgiegpidaaftrincqgktylfkgsqywrfedgvldpdyprnisdgfdgipdn vdaala lpahsysgrervyffkgkqyweyqfqhqpsqeecegsslsavfehfammqrdswedifellfwgrt sagtrqpqfisrdwhgvpgqvdaamagriyisgmaprpslakkqrfrhrnrkgyrsqrghsrgrnq nsrrpsramwlslfsseesnlgannyddyrmdwlvpatcepiqsvfffsgdkyyrvnlrtrrvdtv dppyprsiaqywlgcpapghl (SEQ ID NO: 1)

Gpe protein sequence with PelB signal sequence (NCBI reference sequence: NP_049736.1, protein name: glycoside hydrolase family protein (Escherichia phage T4). PelB signal sequence: 1-22, T4 bacteriophage lysozyme 23-164):

Mkyllptaaagllllaaqpamamnifemlriderlrlkiykdtegyytigighlltkspslnaaks eldkaigrncngvitkdeaeklfnqdvdaavrgilrnaklkpvydsldavrrcalinmvfqmgetg vagftnslrmlqqkrwdeaavnlaksiwynqtpnrakrvittfrtgtwdayknl

Claims (22)

1. A method of producing a purified cell culture comprising a recombinant protein, wherein the method comprises: (i) inoculating the bioreactor with a host cell to provide a cell culture, wherein said host cell is Escherichia coli, a transformed expression system comprising: (a) a nucleic acid encoding a recombinant protein operably linked to a first inducible promoter, wherein the protein is vicronectin (VTN); and b) a nucleic acid encoding a T4 lysozyme protein in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter, ii) fermentation in cell culture under conditions which: (a) enables expression of the recombinant polypeptide; and (b) enables the cell to be permeabilized to form a spheroplast that secretes the recombinant polypeptide; iii) purifying the fermented cell culture comprising the secreted recombinant protein, wherein said purification comprises (a) a nucleic acid inactivation step prior to the pre-clarification step comprising adding a nucleic acid inactivating agent to the cell culture, wherein optionally the nucleic acid inactivating agent is benzonase; and (b) a primary clarification step using a first clarification filter having a pore size of at least about 0.4 μm or greater retention limits, where the clarification step does not include centrifugation. 2. The method of claim 1, further comprising a secondary clarification step using a second filter having a pore size of at least about 0.4 μm or greater retention limits. 3. A method according to claim 1 or 2, characterized in that the primary clarification step uses a first filter having a pore size that provides retention limits of at least about 1.0 to about 3.0 μm. 4. The method of claim 2 or 3, wherein the second clarification stage uses a second filter having a pore size that provides retention limits of at least about 0.22 μm, at least about 1.0 to about 3.0 μm, or at least about 0 .4 μm to about 0.8 μm. 5. A method according to any one of claims 1-4, characterized in that the primary clarification step and/or the secondary clarification step is microfiltration. 6. A method according to claim 5, characterized in that the microfiltration is deep filtration. 7. A method according to claim 5 or 6, characterized in that the microfiltration is selected from normal flow filtration (NFF) or tangential flow filtration (TFF). 8. A method according to any preceding claim, wherein the clarified cell culture has a turbidity of less than about 20 NTU. 9. A method according to any of the preceding claims, characterized in that said fermentation step comprises the addition of a stabilizing agent to the cell culture. 10. A method according to any of the preceding claims, characterized in that the method is carried out in a closed system such that a sterile flow is maintained between the bioreactor and the first and second clarification filters. 11. A method according to any one of the preceding claims, further comprising the step of subjecting said clarified cell culture to one or more purification steps of said recombinant protein. 12. A protein production method comprising: i. culturing a recombinant Escherichia coli cell under conditions that: (a) enables expression of the protein; and (b) allows the cell to become permeable to form a spheroplast that secretes the protein; and ii. isolating the protein from the culture without complete lysis of the recombinant cell, wherein the cell comprises an expression system comprising: (a) a nucleic acid encoding a protein operably linked to a first inducible promoter, wherein the protein is vitronectin (VTN); and (b) a nucleic acid encoding T4 lysozyme in frame with a nucleic acid encoding a PelB secretory signal peptide operably linked to a second inducible promoter. 13. A method according to any one of claims 1-12, characterized in that the method does not include: (i) mechanical cell disruption; (ii) addition of an exogenous enzyme that degrades the cell wall; and (iii) transforming the cell with expression vectors containing a nucleic acid encoding an exogenous enzyme that disrupts the cytoplasmic membrane. 14. The method according to any one of claims 1-13, characterized in that the nucleic acid encoding the protein operably linked to the first inducible promoter is in the first construct and the nucleic acid encoding T4 lysozyme is in frame with the nucleic acid encoding PelB. the secretory signal peptide is present in the second construct. 15. The method according to any one of claims 1-14, characterized in that the first inducible promoter and the second inducible promoter are sequentially induced. 16. The method according to any one of claims 1-14, characterized in that the first inducible promoter and the second inducible promoter are simultaneously induced. 17. The method of any preceding claim, wherein the first inducible promoter is an IPTG-inducible promoter, optionally wherein the IPTG-inducible promoter is PT7. 18. A method according to any of the preceding claims, characterized in that the second inducible promoter is an arabinose-inducible promoter, optionally wherein the arabinose-inducible promoter is araB. 19. The method according to any of the preceding claims, characterized in that the T4 lysozyme enzyme is encoded by the gene E. 20. A method according to any of the preceding claims, characterized in that the E. coli is an Escherichia coli B strain. 21. A method according to any of the preceding claims, further characterized in that the fermentation or culturing comprises changing the first temperature to a second temperature, optionally wherein the second temperature is in the range of 25°C to 36°C and the first temperature is in the range of 36.8° C to 37.2 °C. 22. A recombinant protein produced by the method according to any one of claims 1-21.