MXPA06008061A - Expression of mammalian proteins in pseudomonas fluorescens - Google Patents

Abstract

The invention is a process for improved production of a recombinant mammalian protein by expression in a Pseudomonad, particularly in a Pseudomonas fluorescens organism. The process improves production of mammalian proteins, particularly human or human-derived proteins, over known expression systems such as E. coli in comparable circumstances. Processes for improved production of isolated mammalian, particularly human, proteins are provided.

Description

EXPRESSION OF MAMÍ FERO PROTEINS IN PSEUDOMONAS FLUORESCENS REFERENCES TO PREVIOUS REQUESTS This application claims the priority of United States Provisional Applications Nos. 60 / 564,798, entitled "Expression of mammalian proteins in Pseudomonas fluorescens", filed April 22, 2004, and 60 / 537,148, entitled " Protein expression systems "presented on January 6, 2004.

FIELD OF THE INVENTION The invention is a process for the improved production of a mammalian recombinant protein by expression in a Pseudomonas, particularly in an organism Pseudomonas fluorescens. The process improves the production of mammalian protein expression, over known expression systems.

BACKGROUND OF THE INVENTION More than 325 million people worldwide have been aided by more than 1 55 biotechnology drugs and vaccines approved by the Food and Drug Administration of the Americas.

United States (FDA). In addition, there are more than 370 products and biotechnology pharmacological vaccines, currently in clinical trials addressing more than 200 diseases, including various cancers, Alzheimer's disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis. Contrary to the traditional therapeutics of small molecules that are produced through classical chemical synthesis, proteins are usually produced in living cells inefficiently at a high cost. Due to the high cost of complexity, there is a cut in manufacturing capacity for protein-based therapeutics. The use of microbial cells to produce products has a very long history. As early as 1897, Buchner discovered that enzymes extracted from yeast are effective in converting sugar to alcohol, leading to the production of key initial chemical products using microorganisms. By the time of 1 940, large scale production of pencillin was achieved through fermentation. Techniques for the insertion of foreign genes into bacteria were first developed in the early 1970s. The bacterial production of the commercially viable recombinant mammalian protein was first exploited in the production of human insulin (Goeddel, et al. 1: 179a; Wong, 1992). Nowadays fermentation and cell culture support the large volume of industrial production of alcohol, antibiotics, biochemical products and therapeutic proteins. However, the development and manufacture of therapeutically useful proteins has been impeded due, in large part, to the limitations of the current organisms used to express these endogenous proteins.

Expression of prokaryotic versus eukaryotic proteins Although bacterial expression systems are frequently used to produce recombinant eukaryotic proteins, typically the proteins produced differ significantly from their original counterparts. In general, it is a challenge to produce eukaryotic secondary and tertiary structures in E. coli expression systems. At the same time, while eukaryotic expression systems are currently better at being able to form the secondary and tertiary structures of recombinant eukaryotic proteins, the ability of these systems to produce recombinant proteins in large quantities is limited. The post-translational modifications represent the most significant differences between the expression of prokaryotic and eukaryotic proteins. Prokaryotes (for example, bacteria) have a very simple cell structure and do not have organelles bound to the membrane. In eukaryotes, a protein is frequently modified after it is first produced. These modifications, in many cases, are necessary to convert the peptide to a functional form. In this way, even when existing bacterial expression systems produce a protein with the correct primary structure, the protein may not be post-translationally modified, and is therefore often non-functional. Common modifications include disulfide bond formation, glycosylation, acetylation, acylation, phosphorylation or gamma-carboxylation, all of which can regulate the folding of the protein and the biological activity thereof. Bacterial expression systems generally do not glycosylate, acetylate, acylate, phosphorylate or gamma-carboxylate properly eukaryotic proteins. Bacteria, such as E. coli can form disulfide bonds, but the bonds are often formed in the wrong configuration required for biological activity; therefore, denaturation and refolding are usually required to produce active eukaryotic proteins. Molecular chaperone proteins are present in prokaryotes and eukaryotes that facilitate the folding of other proteins. In the absence of such chaperones, the unfolded or partially folded polypeptide chains are unstable within the cells, frequently folding incorrectly or adding to insoluble complexes. The linkage of the chaperones stabilizes these unfolded polypeptides, thereby preventing incorrect folding or aggregation, and allowing the polypeptide chain to fold in its correct conformation. However, chaperones differ in each cell type, and can be expressed differently based on extracellular conditions.

Problems with current expression systems Escherichia coli (E. coli) is the most widely and routinely used protein expression system. The production in E. coli is cheap, fast and well characterized. In addition, the elevation of scale and harvest is also possible, and the production of cGMP is well established. However, there are significant limitations to the use of E. coli that often prove difficult to overcome, particularly when expressing recombinant mammalian proteins. Along with the limitations described above, high level expression of recombinant gene products in E. coli often results in misfolding of the protein of interest and its subsequent degradation by cellular proteases or deposition within a known biologically inactive aggregate as bodies of inclusion. The protein found in inclusion bodies typically must be extracted and renatured for activity, adding time and expense to the process. Typical methods of renaturation involve attempts to dissolve the aggregate denaturing concentrate, and subsequent removal of the denaturant by dilution. Some of the factors that have been suggested as involved in the formation of inclusion bodies include the high local concentration of the protein; a network environment in the cytoplasm (the cytoplasm E. coli has a broad level of glutathione) preventing the formation of disulfide bonds; the lack of post-translational modifications that can increase the solubility of proteins; Inadequate interactions with chaperones and other enzymes involved in in vivo folding; intermolecular crosslinking via disulfide bonds and other covalent bonds; and the increased aggregation of the folding intermediates, due to their limited solubility. This is probably a combination of its factors, as well as a limited bioavailability of the chaperones, which most commonly leads to the formation of inclusion bodies. Yeast expression formulas, such as Saccharomyces cerevisiae or Pichia pastoris, are also commonly used to produce proteins. These systems are also characterized, provide good levels of expression and are relatively fast and cheap compared to other eukaryotic expression systems. However, the levad uras can achieve only limited post-translational modifications of the protein, the protein may need refolding, and the harvest of the protein may be a problem due to the characteristics of the cell wall. Expression systems in insect cells have also emerged as an attractive but expensive alteive, such as a protein expression system. Well-folded proteins that are generally post-translationally modified, can sometimes be produced, and extracellular expression has been achieved. However, this is not as fast as bacteria and yeast, the rise in scale is generally difficult. Expression systems in mammalian cells, such as Chinese hamster ovary cells, are often used for the expression of complete proteins. This system usually produces correctly folded proteins with the appropriate post-translational modifications and the proteins can be expressed extracellularly. However, the system is very cheap, the scaling is slow and often not feasible, and the yields of the proteins are lower than in any other system.

Pseudomonas fluorescens (P. fluorescens) Pseudomonas fluorescens encompasses a group of common non-pathogenic saprophytes, which colonize soil, water and the plant surface environment, P. fluorescens are expressly used in agricultural and industrial processes, including commercially for the production of non-mammalian industrial and agricultural proteins. Non-mammalian enzymes derived from P. fluorescens have been used to reduce environmental pollution, as detergent additives, and for stereoselective hydrolysis. Mycogen began the expression of recombinant bacterial proteins in P. fluorescens in the mid-1 980s and presented its first patent application on the expression of the Bacillus thuringiensis toxin in P. fluorescens on January 22, 1985 ("cellular encapsulation of biological pesticides "). Between 1 985 and 2004, Mycogen, later Dow Agro Sciences, as well as other companies capitalized on the agricultural use of the peptide P. fluorescens in patent applications on the production of pesticides, insecticides and nematicidal toxins, as well as other specific toxic sequences and the genetic manipulation to improve the expression of these. Examples of patent applications directed to the expression of recombinant bacterial proteins in P. fluorescens include: U.S. Patent Nos. 3,844,893; 3,878,093, 4, 1 69.01 0; 5,292,507; 5,558,862; 5,559.01 5; 5.61 0.044; 5,622,846; ,643,774; 5,662,898; 5,677,127; 5,686,282; 3,844, 893; 3, 878.093; 4,169,010; 5,232,840; 5,292,507; 5,558,862; 5,559,015; 5,610,044; 5,622,846; 5,643,774; 5,662,898; 5,677,127; 5,686,282; 5,686,283; 5,698,425; 5,710,031; 5,728,574; 5,731,280; 5,741,663; 5,756,087; 5,766,926; 5,824,472; 5,869,038; 5,891,688; 5,952,208; 5,955,348; 6,051,383; 6,117,670; 6,184,440; 6,194,194; 6,268,549; 6,277,625; 6,329,172; 6,447,770; as well as PCT Publications Nos. WO 00/15761; WO 00/29604; WO 01/27258; WO 02/068660; WO 02/14551; WO 02/16940; WO 03/089455; WO 04/006657; WO 04/011628; WO 87/05937; WO 87/05938; WO 95/03395; WO 98/24919; WO 99/09834; and WO 99/53035. On October 8, 2003, Dow AgroSciences introduced PCT publication No. 04/087864 entitled, "Amended recombinant cells (ARCs) for the production and distribution of antiviral agents, adjuvants and vaccine accelerators". The application describes the recombinant cells that can include at least one heterologous gene coding for a chemokine or a cytokine and the administration of such cells to a host, to accelerate an immune response. The application demonstrates the production of interferon-a and interferon-? bovines in P. fluorescens. Dow Global Technologies currently has several pending patent applications in the area of the use of P. fluorescens, to produce recombinant proteins. PCT application WO 03/068926 to Dow Global Technologies, filed on February 13, 2003, entitled "Over-expression of extremozyme genes in Pseudomonas and closely related bacteria" describe an expression system in which pseudomonas, specifically P fluorescens, can be used as host cells for the production of extremozine enzymes. These enzymes are typically ancient, found in prokaryotes, eukaryotes, including fungi, yeasts, lichens, protists and protozoa, algae, and mosses, tardigrades and fish. The patent discloses that enzymes can be derived from certain fungi and extremophilic yeasts, but are typically derived from extremophilic bacteria. PCT publication No. WO 03/089455 to Dow Global Technologies, filed on April 22, 2003, entitled "Low cost production of peptides" describes a method for producing small peptides, mainly antimicrobial peptides, as concatameric precursors in pseudomonas, specifically P. fluorescens. PCT publication No. WO 04/005221 to Dow Global Technologies, entitled "Promoters induced by benzoate and anthranilate" provides the new promoters induced by benzoate and anthranilate of P. fluorescens, as well as the new promoters in tandem, variants and improved mutants thereof, which are useful for prokaryotic fermentation systems commercial. U.S. Patent No. 5,232,840 to Monsanto Co., describes the use of the new ribosomal binding sites to increase the expression of certain prokaryotic cell proteins. In one example, the cells are used to express the porcine growth hormone in several organisms including E. coli, P. fluorescens and P. putida. The data show that P. fluorescens is less efficient to express growth hormone when compared to E. coli. In contrast, when expressing a bacterial protein, P. fluorescens is much more effective in the production of proteins than it is. coli under comparable conditions. In fact, the P. fluorescens cells described in this patent produce several times more bacterially derived β-galactosidase from E. coli (compare table 4 to tables 1 and 2). While some progress has been made in the production of proteins of commercial interest, there remains a strong need to improve the capacity and level of production of mammalian recombinant proteins, and in particular human proteins. Therefore, an objective of the present invention is to provide a process for the production of mammalian recombinant proteins, particularly human, which can be isolated and purified for therapeutic use, and the cells that can achieve this process. A further objective of the present invention is to provide improved processes for the production of mammalian recombinant active proteins including mammalian complex proteins. A further objective of the present invention is to provide improved processes for the production of high levels of proteins of recombinant mammals, in particular human proteins. A further objective of the present invention is to provide transformed organisms that provide high levels of soluble and insoluble recombinant mammalian protein expression.

BRIEF DESCRIPTION OF THE INVENTION It has been discovered that Pseudomonas fluorescens is a superior organism for the production of recombinant proteins, and in particular mammalian recombinant proteins, such as recombinant human proteins. Based on these findings, the present invention provides a process for the production of mammalian or mammalian derived recombinant proteins, in P. fluorescens. In addition, the invention provides transformed P. fluorescens to produce mammalian recombinant proteins, including human proteins. In one embodiment, the invention provides a process for producing a mammalian protein in a P. fluorescens organism, in which the protein is produced at a higher level or concentration per cell or per liter fermentation reaction, than in an organism. E. coli under comparable conditions. In yet another embodiment, the invention provides a process for the production of mammalian proteins in a P. fluorescens organism in a batch culture, which produces higher amounts of protein per liter than a corresponding batch of recombinant E. coli organisms. .

Comparable conditions or substantially comparable conditions relate particularly to the expression of the recombinant protein using the same operably linked transcriptional promoter, and the ribosomal binding site in different organisms, and using the same initial induction conditions. The comparable conditions may further include the use of the same vector and associated regulatory elements including, but not limited to, enhancer sequences, termination sequences and origin or replication sequences. The comparable conditions may also include the same total volume of the cell fermentation reaction. The comparable conditions may also include the same concentration of the total cells per liter of reaction. In one embodiment, conditions also include total induction times (before measurement) that are similar or equal. However, in another modality, the induction times may vary depending on the organism. Specifically, P. fluorescens has the capacity for increased growth time on E. coli, without reducting protein production, such that protein production can be measured in P. fluorescens at a point of time at which the cells of E. coli are to a large extent. One way to measure comparable conditions is to compare the percentage of recombinant protein with total cellular protein. The comparable conditions also do not require identical means for development. The media can be adjusted to ensure optimal production for individual organisms.

In yet another embodiment, the invention provides a process for producing mammalian recombinant proteins by producing the proteins in an organism of P. fluorescens and isolating the protein produced. In a secondary embodiment, the process includes substantially purifying the protein. In one embodiment, the protein is derived from a human protein, or is humanized. The invention also provides the use of P. fluorescens in at least the following embodiments: (i) the production of mammalian recombinant proteins, including human, present in the cell in a range of 1 and 75 percent of the total cellular protein ( % tcp), or in particular, at least more than about 5% tcp, 1 0% tcp, at least 1 5% tcp, at least 20% tcp or more; (ii) the production of recombinant mammalian proteins, including human ones, which are soluble and are present in the cytoplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 1 0% tcp, at least 1 5% tcp, at least 20% tcp or more; (iii) the production of mammalian recombinant proteins, including human, which are insoluble in the cytoplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 10% tcp , at least 1 5% tcp, at least 20% tcp or more; (iv) the production of recombinant mammalian proteins, including human, which are soluble in the periplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 10% tcp, at least 15% tcp, at least 20% tcp or more; (v) the production of mammalian recombinant proteins, including human, which are insoluble in the periplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 10% tcp, at least 1 5% tcp, at least 20% tcp or more; (vi) the production of mammalian recombinant proteins, including human, in the cell in a range between 1 and 75% tcp, or particularly at least more than about 5% tcp, 10% tcp, at least 15% tcp , at least 20% tcp or more, when developed at a cell density of at least 40 g / l; (vii) the production of recombinant mammalian proteins, including human, present in the cell in an active form; (viii) the production of recombinant mammalian proteins, including human, multi-subunit, in active form; (ix) the production of recombinant mammalian proteins, including human proteins, which are then isolated and purified; and (x) the production of recombinant mammalian proteins, including humans, which are renatured. In one embodiment, the recombinant mammalian protein is selected from the group consisting of a multi-subunit protein, a blood carrier protein, an enzyme, a full-length antibody, an antibody fragment or a transcriptional factor. In a further embodiment, the invention includes: (i) Pseudomonas fluorescens organisms that are transformed to produce recombinant mammalian proteins, including human, at a higher level or concentration than an organism of E. coli corresponding, when it develops under substantially corresponding conditions; (ii) Organisms of Pseudomonas fluorescens that are transformed to produce mammalian recombinant proteins, including human and peptides that are present in the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 1 0% tcp, at least 1 5% tcp, at least 20% tcp or more; (iii) Organisms of Pseudomonas fluorescens that are transformed to produce recombinant mammalian proteins, including humans that are present in the cell in active form; (iv) Organisms of Pseudomonas fluorescens that are transformed to produce mammalian recombinant proteins, including humans that are soluble in the cytoplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 1 0% tcp, at least '1 5% tcp, at least 20% tcp or more;; (v) Organisms of Pseudomonas fluorescens that are transformed to produce mammalian recombinant proteins, including human that are insoluble in the cytoplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp, 1 0% tcp, at least 1 5% tcp, at least % tcp or more;; (vi) Organisms of Pseudomonas fluorescens that are transformed to produce mammalian recombinant proteins, including humans that are soluble in the periplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp , 1 0% tcp, at least 1 5% tcp, at least % tcp or more;; (vii) Organisms of Pseudomonas fluorescens that are transformed to produce recombinant mammalian proteins, including humans that are insoluble in the periplasm of the cell in a range of between 1 and 75% ctp or in particular at least more than about 5% tcp , 1 0% tcp, at least 1 5% tcp, at least 20% tcp or more;; (viii) Pseudomonas fluorescens organisms that are transformed to produce recombinant mammalian proteins, including human, multisubunit; (ix) Organisms of Pseudomonas fluorescens that are transformed to produce mammalian recombinant proteins, including human multisubunit, present in the cell in active form. In an alternative embodiment, the Pseudomonas organisms and closely related bacteria other than fluorescens are used as host cells in this invention, as described in more detail below. In one embodiment, the host cell will be selected in general from the genus Pseudomonas and specifically from a non-pathogenic Pseudomonas species. Likewise, any strain of Pseudomonas fluorescens can be used, which achieves the desired inventive goal, including but not limited to strain MB1 01, or a strain that is modified to include at least one inserted copy, expressible in the host cell, of at least one Lac transgene encoding the Lacl repressor protein, such as MB214 and MB21 7. The organism Pseudomonas may also be optionally genetically modified to add or delete one or more genes, to improve the functioning, processing or other characteristics. In one embodiment, the Pseudomonas organism is transformed with a nucleic acid encoding a mammalian recombinant protein selected from the group consisting of a multi-subunit protein, a single-stranded carrier protein, a full-length antibody, an antibody fragment, or a transcriptional factor. In one embodiment, the organism Pseudomonas fluorescens expresses a mammalian recombinant protein selected from the group consisting of a multi-subunit protein, a blood carrier protein, an enzyme, a full-length antibody, an antibody fragment or a transcriptional factor. The recombinant mammalian or human protein, expressed will typically have a mass of at least about 1 kD, and up to about 1, 200, 300, 400 or 500 kD, often between about 10 kD and about 1000 kD, and usually more than about 30 kD.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing purified hu -? - I FN from the soluble fraction of P. fluorescens samples showing activity comparable to a commercially available standard. Figure 2 is a graph of an ELISA assay showing the activity of Gal 1 3 encoded with P. fluorescens and E. coli. Figure 3 depicts expression constructs of human growth hormone. The amino acid sequence of the human growth hormone lacking its native secretion signal sequence is shown in A. The plasmid constructs for expression in P. fluorescens (pDOW2400) and E. coli (41 2-001. HGH ) are shown in B. Figure 4 is an image of an S DS-PAGE analysis of soluble and insoluble fractions of hGH expressed in P. fluorescens and E. coli. The post-induction in time is denoted by 10, I24, I48, 0 or 3. The large arrows indicate the position of the hG H protein of 21 kDa. Figure 5 shows an SDS-PAGE analysis of the expression of? -I FN in E. coli versus P. fluorescens cells. The soluble (S) and insoluble fractions (I) of the samples taken at 0, 3, 4 and 48 hours after the induction (10, etc., were resolved). ? -I FN expressed in E. coli is shown in panel A,? -I FN expressed in P. fluorescens is shown in panel B. Samples of 5 μL of A575-20 were loaded onto a Bis-Tris NuPAGE gel at 1 0% and resolved in a 1 X MONTH. The arrows indicate the position of the recombinant protein. Western analyzes are shown in panels C. (E. coli) and D (P. fluorescens). Figure 6 shows the replacement of the BuiBui toxin gene with the BGI gene in the Spel and Xhol sites of pMYC 1 803. Figure 7 shows that all selected transformants had the desired interferon insert, as verified by DNA sequencing inserted. Figure 8 depicts the nucleotide sequence for the protein fusion single chain antibody phosphate-binding protein ga! 2. Figure 9 depicts the amino acid sequence for the protein fusion single chain phosphate-binding antibody gal2. Figure 10 represents the nucleotide sequence for the fusion protein phosphate-binding human growth hormone protein. Figure 11 represents the amino acid sequence for the phosphate-human growth hormone binding protein fusion protein.

DETAILED DESCRIPTION OF THE INVENTION Process for Producing Mammalian Recombinant Proteins The invention provides the processes and transformed organisms of Pseudomonas fluorescens that produce mammalian recombinant proteins. In one embodiment, the invention provides a process for producing mammalian recombinant proteins by producing the proteins in a P. fluorescens organism, and isolating the produced protein. The protein can be isolated after expression by techniques known in the art, including but not limited to affinity chromatography, ion exchange chromatography, antibody affinity, size exclusion and any other method that removes a substantial portion of the waste. cellular protein. In a secondary embodiment, the process provides a substantially purified protein. The isolated protein may have activity similar to that of the native protein from which it is derived. The protein can be isolated in a correctly folded state or conformation, approaching aq ue of the native protein, which can be further renatured or modified to put it into a properly folded conformation. In a secondary mode, the protein is derived from a human protein, or is humanized. A "humanized" protein is typically a chimeric mammalian protein that is partially comprised of a human-derived protein sequence. Humanization is partially useful in the production of antibodies and the development of humanized antibodies has been extensively described, for example, in U.S. Patent No. 6,800,738. In one embodiment, the expression of the protein by the host cell is followed by the isolation of the protein. In yet another embodiment, the peptide protein is purified. In an alternative embodiment, the protein is purified after isolation of the protein. Optionally, the isolated and purified protein can be renatured or refolded in order to produce active proteins. In a further embodiment, the invention provides a process for producing a mammalian protein in an organism of P. fluorescens in which the protein is produced at a higher level or concentration than in an E. coli organism. The adequacy of P. fluorescens organisms for the high level production of mammalian proteins was unexpected, based on the lack of success in the production of such proteins in those organisms in the prior art. The present inventors have found that these organisms are rather capable of having a high level of production of mammalian proteins, and typically express the protein in a higher yield or at higher levels than E. coli when tested in corresponding assays. . In a further embodiment, the invention provides a process for producing mammalian proteins in an organism of P. fluorescens in a batch culture, which produces higher amounts of protein per liter than a corresponding batch of recombinant E. coli organisms. . In some embodiments, processes that include the production of recombinant mammalian multi-subunit proteins, including human, in active form in P. fluorescens are provided.; the production of mammalian blood carrier proteins, recombinants, including human blood carrier proteins such as transferin and albumin in P. fluorescens, the production of mammalian recombinant enzymes, including recombinant mammalian enzymes in active form in P. fluorescens; the production of antibodies and antibody fragments including single chain antibodies and Fab fragments in P. fluorescens; and the production of mammalian recombinant transcriptional factors, including humans, in P. fluorescens. In one embodiment, the mammalian recombinant protein is produced as a multimer, or in a concatamer precursor, for example, in the form of at least two small peptide units (of 1 -15 amino acids) in tandem. In an alternative embodiment, the mammalian recombinant protein is not produced as a multimer, or in precursors with concatamerics, but rather is produced as a single chain polypeptide.

Selection of biomolecules A separate embodiment of the present invention provides P. fluorescens organisms in a mammalian biomolecule libraries selection process, to identify at least one desired activity or property. P. fluorescens cells can be transformed with a number of mammalian derived nucleic acids for which the test is desired, the production of a library of transformed host cells. After expression, the polypeptides encoded by at least some of the nucleic acids are produced to be tested either in the cytoplasm or after recovery of the cell. Examples of activities and properties for which the test may be performed include: level of expression of the polypeptide; stability of the polypeptide; activities and biocatilitic properties. Illustrative examples of biocatalytic activities and properties include: enzymatic activities; interactions / protein link; protein stabilization; use of the substrate; product formation; reaction conditions, such as pH, salinity, or reaction temperature; biocatalytic parameters for a given catalysed reaction, such as Km and Vmax, and stability behavior, such as thermostability and biocatalyst half-life. The obtained test results can be used to select one or several members of the library for further development.

Protein Expression A key aspect of this invention is the expression of high levels of mammalian recombinant proteins, eg, human proteins, in a range of between 1 and 75 percent of the total cellular protein (% tcp) by expression in organism of P. fluorescens. The expressed proteins can be soluble or insoluble, while they are in the cell of P. fluorescens. Such high levels of soluble or insoluble mammalian recombinant proteins may be an improvement over previously known mammalian protein expression systems. In particular, high levels of mammalian proteins recovered in large-scale fermentation reactions are typically not possible as known techniques. In one embodiment, the invention provides expression levels of mammalian proteins that exceed those found in E. coli expression systems. In one embodiment, the concentration of the recombinant proteins in each cell is higher than that found in E. coli in comparative tests. In one embodiment, the level of recombinant protein compared to total cellular cellular protein in the expression system of P. fluorescens is higher than that of the same recombinant protein expressed in E. coli. In yet another embodiment, the level of the amount of the soluble protein in the expression system of P. fluorescens described herein, is higher than the level or amount of soluble recombinant protein in a comparable E. coli expression system. . In yet another embodiment, the total amount of the active protein is greater than the amount derived from an expression system of £. coli In a separate embodiment, the level of recombinant active protein compared to the total cellular protein measured in the expression system of P. fluorescens is higher than that of the same recombinant protein expressed in E. coli. In one embodiment, the level, concentration, or amounts of proteins expressed in P. fluorescens is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least at least 9x, at least 10x, or more of the level, concentration or amount of the recombinant protein expressed in E. coli in comparable assays. One of the benefits of P. fluorescens as an expression system is that the cells can be grown in large-scale cultures without negatively impacting their capacity for protein production. This capacity exceeds those found in other bacterial systems, such as E. coli. In another embodiment, the process includes the production of mammalian proteins in batch cultures in which the recombinant protein is produced at a higher total level in P. fluorescens than in the batch cultures of E. coli. In yet another embodiment, the invention provides a process for producing mammalian proteins in a P. fluorescens organism in a batch culture, which produces higher amounts of protein per liter than a corresponding batch of recombinant E. coli organisms. . The invention generally provides processes and transformed organisms of P. fluorescens that provide expression levels of 1-75% of the total cellular protein (tcp) of soluble and insoluble recombinant mammalian proteins. Recombinant mammalian proteins expressed in the cell can be expressed in an active form. In other embodiments, P. fluorescens provides at least 1, 5, 10, 15, 20, 25, 30, 40, 50, 55, 60, 65, 70, or 75% tcp of recombinant mammalian proteins. These proteins can be soluble, and when they are soluble, they can be present in the cytoplasm or in the periplasm of the cells during production. Soluble proteins are readily released from the cell by methods including, but not limited to, rupture of the cell membrane by pressure (for example, the "French" press method) or by degradation with lysozyme of the cell membrane. The cells can also be typically lysed using detergents, such as non-ionic detergents. Proteins that are soluble can also be stabilized by adjusting the components of the buffer, such as buffer pH, salt concentration or additional protein components (eg, in multi-subunit complexes). Soluble proteins can be isolated or purified from another protein and cellular debris by, for example, centrifugation and / or chromatography, such as size exclusion chromatography, anionic or cationic or affinity exchange chromatography. Proteins can also be insoluble, insoluble proteins are typically found in inclusion bodies of the cytoplasm, but they are also frequently in the periplasm. Not all insoluble proteins are in inclusion bodies, and they can also be found in membrane aggregates, as small protein aggregates or in any other insoluble form in the cytoplasm or in the periplasm. Insoluble proteins can typically be renatured using, for example, reductive agents such as urea or guanidine hydrochloride. Insoluble proteins or protein aggregates can be isolated, for example, by centrifugation and / or chromatography such as size exclusion chromatography. Proteins in insoluble aggregates can typically be separated by solubilizing the aggregates using, for example, micelles or reverse micelles as described in Vinogradov, et al. (2003) Anal Biochem. 1 5: 320 (2): 234-8. In a particular embodiment, the host cell of Pseudomonas may have a recombinant mammalian peptide, polypeptide, protein or fragment thereof, with an expression level of at least 1% tcp and a cell density of at least 40 g / liter, when developed (for example, within a range of about 4 ° C to about 55 ° C, inclusive) in a medium of mineral salts. In a particular embodiment, the expression system will have a recombinant protein of the peptide, including the mammalian recombinant protein, expression level of 5% tcp and a cell density of at least 40 g / liter, when developed (e.g., within from a temperature range of about 4 ° C to about 55 ° C, inclusive) in a medium of mineral salts at a fermentation scale of at least 10 liters. Expression levels can be measured by standard techniques known in the art. In a modality, the amount of protein (in grams) is compared to the amount in grams of the total cellular protein in a given sample. In yet another embodiment, the measurement is a level of recombinant protein per liter. In yet another embodiment, the level or quantity can be measured in comparison to a known standard, such as a BSA control. The level or amount of the recombinant modality can be measured, for example, by analyzing the absorption of light from a purified protein, by measuring an affinity of a marker for the protein (such as an antibody affinity) and comparing this to a known standard, or by measuring the level of activity compared to a known standard (such as the known amount of active, purified protein). It has been found that, in certain situations, additional conditions or agents promoting the disulfide bonds are not required, in order to recover the target polypeptides containing disulfide bonds, in soluble and active form, when a Pseudomonas fluorescens bacterium is used as a the host cell of expression. Therefore, in one embodiment, the peptide, polypeptide, protein or transgenic fragment thereof contains at least one intramolecular disulfide bond in its native state. In other embodiments, the protein may contain up to 2, 4, 6, 8, 10, 14, 14, 16, 20 or more disulfide bonds in their native state. In some embodiments, the protein is expressed or found in the periplasm of the cell during production prior to purification or isolation. The protein can be secreted into the periplasm by being fused to an appropriate signal secretion sequence. In one embodiment, the signal sequence is a signal sequence that is native to the P. fluorescens genome. In specific embodiments, the signal sequence is a phosphate binding protein, a secretion signal peptide of the Lys-Arg-Orn binding protein (LAObp or KRObp), a Membrane Porin E secretion signal peptide. Exterior (OpreE), an azurine secretion signal peptide, a signal peptide secretion of the iron binding protein (III) (Fe (III) bp), or a signal peptide secreting lipoprotein B ( LprB). In one embodiment, the recombinant peptide, polypeptide, protein or fragment thereof has a folded intramolecular conformation in its active state. P. fluorescens typically produces mammalian proteins more efficiently in correctly folded conformation. In one embodiment, more than 50% of the transgenic peptide, the polypeptide, the protein or the expressed fragment thereof produced, can be produced as single peptides, polypeptides, proteins, or fragments thereof in soluble and active form or in insoluble form , but renaturalizable in the cytoplasm or periplasm. In other embodiments, approximately 60%, 70%, 75%, 80%, 85%, 90%, 95% of the expressed protein is obtained in or can be renatured in active form. Definitions Throughout this specification, the term "protein" is used to include any concatamers or amino acid polymers. The terms "polypeptide", "peptide" and "protein" are used interchangeably and include the polymers of amino acids in which one or more amino acid residues is an artificial chemical analog of a corresponding amino acid of natural origin, as well as the polymers of amino acids of natural origin. The term "isolated" refers to the nucleic acid, the protein or peptide that is substantially or essentially free of other material components that normally accompany it, as they are found in their native state when they are in a cell, eg, other cellular components . The term "purified" or "substantially purified" is used to mean that the protein is separated from other cellular components and is separated from other proteins and peptides found in the cell that are not in a complex native to the protein. In particular modalities, the purified proteins are of an approved purity for therapeutic or veterinary use, as defined by standard cGMP guidelines or approved by the FDA. The term "total cell protein percentage" ("tcp") means the amount of protein in the host cell as a percentage of the aggregated cellular protein.Alternatively, the term means a measure of the fraction of the total cellular protein representing the amount relative to a given protein expressed by the cell The term "operably linked or linked" refers to any configuration in which the transcriptional and translational regulatory elements are covalently linked to the coding sequence in such or such arrangements, relative to the coding sequence, which in and by action of the host cell, the regulatory elements can direct the expression of the coding sequence As used herein, the term "mammalian" is understood to include or designate any animal in the mammary class including human or non-human mammals, such as, but not limited to, porcine s, sheep, cattle, rodents, ungulates, pigs, sheep, lambs, goats, cattle, deer, mules, horses, donkeys, monkeys, dogs, cats, rats and mice. As used herein, the term "mammalian recombinant protein" or peptide is understood to include proteins derived from a native mammalian protein sequence, or derived, or generated from a native mammalian nucleic acid sequence. . Such recombinant proteins can be produced from the nucleic acid sequences which correspond substantially to the mammalian mRNA, native or to the correspondingly corresponding cDNA, or fragments thereof. The sequence can be adjusted accordingly based on the use of the specific host cell codon, as is known in the art. The phrase "substantially corresponding" in the context of two nucleic acids or polypeptides refers to the residues in the two sequences having at least 50%, 60%, 70%, 80%, 90% or greater identity when aligned for the maximum correspondence on a protein domain, as measured using an algorithm known in the art. Optimal alignment of the sequence for comparison can be conducted, for example, by algorithms known in the art (e.g., Smith &; Waterman (1 981) Adv. ' Appl. Math. 2: 482; Needleman and Wunsch (1 970) J. Mol. Biol. 48: 443 Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444; Altschul et al. (1 990) J. Mol. Biol. 21 5: 403-41 0 (BLAST), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), Or by inspection. Software to review BLAST analyzes is only available through the National Center for Biotechnology Information (http: // www. Ncbi. Nlm. Nih. Gov /). The term "fragment" means a portion or partial sequence of a nucleotide, protein or peptide sequence. As used herein, the term "soluble" means that the protein is not precipitated by centrifugation at a gravity between about 5,000x and 20,000x when centrifuged for 10-30 minutes in a buffer under physiological conditions. Soluble proteins are not part of an inclusion body or other precipitated mass. As used herein, the term "insoluble" means that the protein can be precipitated by centrifugation at a gravity between 5,000x and 20,000x when centrifuged for 10-30 minutes in a buffer under physiological conditions. Insoluble proteins can be part of an inclusion body or other precipitated mass. The term "inclusion body" means that it includes any intracellular body contained within a cell, where an aggregate of proteins has been sequestered. As used herein, the term "homologous or homologous" means either i) a protein having an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, or 98% similar to the sequence of a given original protein, and which retains a desired function of the original protein, or ii) a nucleic acid having a sequence that is at least 70, 75, 80, 5, 90, 95, or 98% similar to the sequence of a given nucleic acid, and which retains a desired function, of the original nucleic acid sequence. In all embodiments of this invention of the disclosure any protein, peptide or nucleic acid described can be substituted with a homologous or substantially homologous protein, peptide or nucleic acid which concentrates a desired function. In all embodiments of this invention, and description when any nucleic acid is described, it should be assumed that the invention also includes all nucleic acids that hybridize to the nucleic acid described. In a non-limiting mode, the non-identical amino acid sequence of the homologous polypeptide can be amino acids that are members of any of the conservative or semi-conservative groups shown in Table 1.

TABLE 1: Substitution groups of similar amino acids.

Types of mammalian proteins produced In general, the mammalian recombinant protein can be any mammalian protein of which an amino acid sequence or any putative mammalian or mammalian protein for which an amino acid sequence is derived is known. The proteins can be selected from the group consisting of a multi-subunit protein, a blood carrier protein, an enzyme, a full length antibody, an antibody fragment or a transcriptional factor. The amino acid sequence of the protein can be altered to conform to the desired qualities, such as to ensure certain types of interactions. The sequences may, for example, be adjusted to reduce immunoreactivity, or to increase absorption, reduce excretion, or otherwise increase bioavailability in an organism such as a mammal. The amino acid sequence of the protein can thus be adjusted to ensure certain post-translational modifications or protein conformations. In one embodiment, the mammalian protein is a chemokine or cytokine. In another embodiment, mammalian proteins are one of the following proteins: IL-1, IL-1a, L-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12elasti, IL-13, IL-15, IL-16, IL-18, IL-18BPa, IL-23 , IL-24, VIP, erythropoietin, GM-CSF, G-CSF, M-CSF, platelet-derived growth factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growth factor (FGF; example, aFGF (FGF-1), bFGF (FGF-2), FGF-3, FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors (eg, IGF- 1, IGF-2); tumor necrosis factors (e.g., TNF, lymphotoxin), nerve growth factors (e.g., NGF), vascular endoteiial growth factor (VEGF); Interferons (for example, IFN-a, IFN-β, IFN-β); leukemia inhibitory factor (LIF); ciliary neurotrophic factor (CNTF); oncostatin M; stem or pluripotent stem (SCF) factor; transforming growth factor (e.g., TGF-α, TGF-β1, TGF-β1, TGF-β1); TNF superfamily (eg, LIGHT / TNFSF14, STALL-1 / TNFSF13B (BLy5, BAFF, THANK), TNFalpha / TNFSF2 and TWEAK / TNFSF12), or chemokines (BCA-1 / BLC-1, BRAK / Kec, CXCL16, CXCR3, ENA-78 / LIX, Eotaxin-1, Eotaxin-2 / MPIF-2, Exodus-2 / SLC, Fractalcin / Neurotactin, GROalfa / MGSA, HCC-1, l-TAC, lymphotactin / ATAC / SCM, MCP- 1 / MCAF, MCP-3, MPC-4, MDC / STCP-1 / ABDC-1, MIP-1a, MIB-1β, MIP-2a / GROß, MIPC-3a / Exodus / LARC, MIPC-3a / Exodus / ELC, MIP-4 / PARC / DC-CK1, PF-4, RANTES, SDF1a, TARC, or TECK). Alternatively, the protein is not a chemokine or cytokine. In yet another embodiment, the mammalian protein is not one of the following proteins: IL-1, IL-1a, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12elasti, IL-13, IL-15, IL-16, IL-18, IL-18BPa, IL-23 , IL-24, VIP, erythropoiectin, GM-CSF, G-CSF, M-CSF, platelet-derived growth factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growth factor (FGF; example, aFGF (FGF-1), bFGF (FGF-2), FGF-3, FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors (eg, IGF- 1, IGF-2); tumor necrosis factors (e.g., TNF, lymphotoxin), nerve growth factors (e.g., NGF), vascular endothelial growth factor (VEGF); interferons (for example, IFN-a, IFN-β, IFN-β); leukemia inhibitory factor (Ll F); ciliary neurotrophic factor (CNTF); oncostatin M; stem or pluripotent stem (SCF) factor; transforming growth factors (e.g., TGF-α, TGF-β1, TGF-β1, TGF-β1); TNF superfamily (eg, LIGHT / TNFSF14, STALL-1 / TNFSF13B (BLy5, BAFF, THANK), TNFalpha / TNFSF2 and TWEAK / TNFSF12), or chemokines (BCA-1 / BLC-1, BRAK / Kec, CXCL16, CXR3, ENA-78 / LIX, Eotaxin-1, Eotaxin-2 / MPlF-2, Exodus-2 / SLC, Fractalcin / Neurotactin, GROalfa / MGSA, HCC-1, l-TAC, lymphotactin / ATAC / SCM, MCP-1 / MCAF, MCP-3, MPC-4, MDC / STCP-1 / ABDC-1, MIP-1a, MIB-1β , MIP-2a / GROß, MlPC-3a / Exodus / LARC, MIPC-3 / Exodus / ELC, MIP-4 / PARC / DC-CK1, PF-4, RANTES, SDF1a, TARC, or TECK). In one embodiment, the protein is not a porcine protein, particularly not a porcine growth factor. As a further embodiment of the present disclosure, mammalian recombinant proteins, fragments or derivatives thereof, or analogs thereof, may be antibodies. These antibodies can be, for example, polyclonal or monoclonal antibodies. This aspect of the present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments or the product of a Fab expression library.

Production of multi-subunit proteins In one embodiment of the present invention, the production of multi-subunit mammalian proteins, recombinant, or a host cell of the Pseudomonas species is provided. In yet another embodiment, a host cell of the Pseudomonas species that has been transformed to express a multi-subunit mammalian recombinant protein is provided. In one embodiment, the multi-subunit proteins, including mammalian recombinant proteins or human proteins, are expressed in a Pseudomonas host cell. In one embodiment, expression of the multi-subunit protein by the host cell is followed by isolation of the multi-subunit protein. In yet another embodiment, the multi-subunit protein of the peptide is purified. The protein can be assembled by the cell before purification or isolation, or additional assembly can be undertaken during or after isolation or purification. Optionally, the protein or any portion thereof can be renatured or refolded to produce active proteins. Any of a variety of vectors and expression systems can be used to express the multi-subunit protein in the host cell. The multi-subunits can be located on a simple vector, optionally operably linked to different promoters, optionally in a polycistronic sequence. Each subunit can also be on different vectors. Multiple vectors can be used. Each subunit may be under the control of one or more selection markers. Regulatory elements can be included on the vector, including the periplasmic secretion signal sequences, internal ribosome entry sites, activating sequences, promoters and termination signals. In one embodiment, the multi-subunit proteins are expressed in Pseudomonas using expression systems with auxotrophic selection markers as described in U.S. Application No. 1 0/994, 1 38 to Dow Global Technologies presented on Jan. 9, 1999. November 2004, where the control of each nucleic acid encoding a subunit is under the control of an auxotrophic selection marker. The multi-subunit proteins that can be expressed include homomeric and heteromeric proteins. The multi-subunit proteins may include two or more subunits, which may be the same or different. For example, the protein can be a homomeric protein comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or more subunits. The protein can also be a heteromeric protein that includes 2, 3, 4, 5, 6, 7, 8, 9, 11, 11, or more subunits.

Exemplary multi-subunit mammalian proteins include: receptors that include ion channel receptors; signaling proteins such as kinases, GTPases, ATases; transmembrane proteins; extracellular matrix proteins that include chondroitin; collagen; immunomodulators that include MHC proteins, full-chain antibodies, and antibody fragments; enzymes that include RNA polymerases, and DNA polymerases; and membrane proteins.

Production of blood proteins In one embodiment of the present invention, the production of recombinant mammalian blood proteins is provided. In one embodiment, the expression of the blood protein by the host cell is followed by the isolation of the blood protein. In yet another embodiment, the blood protein is purified. In yet another embodiment, after isolation of the blood protein, the blood protein is purified. Optionally, the protein can be renatured or refolded to produce the active protein. In general, a recombinant blood protein of this invention is produced by transforming a suitable host cell, such as a host cell of P. fluorescens., with a nucleic acid construct encoding the blood protein, culturing the transformed host cell under conditions suitable for expression and optionally isolating, or isolating and purifying the recombinant blood protein expressed by the cell. In yet another embodiment, a host cell of the Pseudomonas species is provided, which has been transformed to express a recombinant mammalian blood protein with a vector containing appropriate genes and regulatory elements appropriate for the expression of the blood protein of interest. . Blood proteins that can be expressed include, but are not limited to: carrier proteins, such as albumin, including human albumin (SEQ ID I No. 1, table 2) and bovine albumin; transferrin, including human transferrin (SEQ ID No. 2, Table 2), bovine transferrin, rat transferrin, recombinant transferrin, recombinant transferrin medians, recombinant transferrin medians that have altered properties, haptoglobin; fibrinogen and other coagulation factors; component of the complement; immunoglobulins, enzyme inhibitors; precursors of substances such as angiotensin and bradykinin; insulin; endothelin; globulin, including alpha, -, beta-; and gamma-globulin and other types of proteins, peptides and fragments thereof, found mainly in the blood of mammals. Amino acid sequences for numerous blood proteins have been reported (see SS Baldwin (1993) Comp. Biochem Physiol. 1 06b: 203-21 8), including the amino acid sequence for human serum albumin (Lawn, LM, et al. (1 981) Nucleic Acids Research 9: 22; pp 6103-61 14) and human serum transferrin (Yang, F., Et al. (1984) Proc. Nati, Acad. Sci. USA 81, pp. 2752-2756) .

In a specific embodiment, albumin production of P. fluorescens is provided which comprises transforming the host cell of P. fluorescens with an expression vector containing a nucleic acid sequence or regulatory sequences and elements for the expression of albumin. , cultivating the host cell under conditions suitable for the expression of albumin, and recovering the albumin expressed by P. fluorescens. According to this embodiment, the expressed albumin is selected from the group consisting of human albumin, bovine albumin, rabbit albumin, chicken albumin, rat albumin, and mouse albumin. In yet another embodiment, the albumin can be fused to a therapeutically active polypeptide, which may be a mammalian or non-mammalian origin. In a further specific embodiment, the production of a transferrin in P. fluorescens is provided, comprising transforming the host cell of P. fluorescens with an expression vector containing the nucleic acid and regulatory elements for the expression of transferrin, the culture of the host cell under conditions suitable for the expression of transferrin. In another embodiment, after the expression of transferrin and, in one embodiment, the isolation of the protein. In a further embodiment, the transferrin can be purified after isolation. The expressed transferrin is selected from the group consisting of human serum transferrin, glycosylated human transferrin, non-glycosylated human transferrin, N-terminal half molecule of human transferrin, bovine transferrin, rat transferrin, mouse transferrin, primate transferrin, recombinant transferrin. , half-molecules of recombinant transferrin, half-molecules of recombinant transferrin having altered properties, transferrin polynucleotides, transferrin polypeptides encoded by transferrin polypeptides, transferrin polypeptides, transferrin antibodies, transferrin fragments, and transferrin fused to a polypeptide therapeutically active. In another specific embodiment, the production of a globulin in P. fluorescens is provided, comprising transforming a host cell of P. fluorescens with an expression vector containing the nucleic acid and regulatory elements for the expression of globulin, culturing the host cell under conditions suitable for the expression of the globulin and optionally isolating the protein. In a further embodiment, after expression, the globulin is isolated and purified from the host cell. The expressed globulin is selected from the group consisting of human globulin, bovine globulin, rabbit globulin, rat globulin, mouse globulin, sheep globulin, monkey globulin, steroid binding globulin, and globulin fused to a therapeutically active polypeptide . In a further embodiment, the production of an insulin in P. fluorescens is provided which comprises the transformation of a P. fluorescens host cell with an expression vector containing nucleic acid and regulatory elements for the expression of insulin, the culture of the host cell under conditions suitable for the expression of insulin and optionally isolating the protein. In a further embodiment, the insulin can be isolated and purified after the production of the insulin by the host cell. The expressed insulin is selected from the group consisting of human insulin, bovine insulin, mouse insulin, rat insulin, porcine insulin, monkey insulin, and insulin fused to a therapeutically active polypeptide. The access number for the human insulin genes is J00265, and for the insulin gene a synthetic number the access number is J02547. Full-length DNA for the production of recombinant blood proteins or truncated DNA encoding either the ammino-terminal or carboxyl-terminal lobe of the blood proteins or a portion thereof, can be obtained from the available sources, or it can be synthesized according to the known sequences, by standard procedures.

TABLE 2. Blood protein sequences expressed by the system of the present disclosure Sequence SEQ MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE of No .: 1 ENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE amino acid PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL s ai KKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKA serum albumin ACLLPKLD EGKASSAKQRLKCASLQKFG ELRD human ERAFKAW AVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECA DDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEY ARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKV FDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYT KVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDY LSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALE VDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELV KHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEE GKKLVAASQAALGL (Lawn, et al. (1981) Nuc.Ac.Rsch.9 (22): 6103-6114) SEQ MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKC No. 2 QSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADA VTLDAGLVYDAYLAPNNLKPWAEFYGSKEDPQTFYYAV AWKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYC s amino acid of Transferrin DLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGC GCSTLNQYFGYSGAFKCLKNGAGDVAFVKHSTIFENLAN KADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTWARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLF KDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCQEA PTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAET TEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENY NKSDNCEDTPEAGYFAVAWKKSASDLTWDNLKGKKSC HTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGD VAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGT Enzyme production In one embodiment of the present invention, the production of mammalian recombinant enzymes or co-factors is provided by a host cell of the species Pseudomonas fluorescens. In yet another embodiment, a host cell of the Pseudomonas species that has been transformed to express a mammalian recombinant enzyme or co-factor is provided. Enzymes and co-factors expressed in this embodiment, include, but are not limited to, aldolases, amine oxidases, amino acid oxidases, aspartases, B12 dependent enzymes, carboxypeptidases, carboxiesterases, chymotrypsin, enzymes requiring CoA, cyanohydrin synthetases , cystathione-synthases, decarboxylases, dehydrogenases, alcoholic dehydrogenases, dehydratases, diaphorases, dioxygenases, enoato-reductases, epoxide-hydrases, fumoreses, galactoses-oxidases, glucose-isomerases, glucose-oxidases, glucosyltransferases, methyltranferases, nitrile-hydrases, nucleoside- phosphorylases, oxidoreductase, oxinitrilases, peptidases, glycosyltranferases, peroxidases, and enzymes fused to a therapeutically active polypeptide. In yet another embodiment, the enzyme may be a mesophilic enzyme polypeptide, for example, one that is desirable for therapeutic and / or human and / or veterinary diagnostic use. Examples of such mesophilic therapeutic enzymes include, for example, tissue plasminogen activator; eurocinase, reptilase, streptokinase, catalase, superoxide dismutase; DNase, amino acid hydrolases (eg, asparaginase, amidohydrolases); carboxypeptidases, proteases, trypsin, pepsin, chymotrypsin, papain, bromelain, collagenase; neuraminidase; lactase, maltase, sucrase and arabinofuranosidases. Yet another embodiment provides for the production of recombinant enzyme replacement in P. fluorescens cells, by transforming a host cell of P. fluorescens, with an expression vector containing nucleic acids and regulatory elements for the expression of the replacements of recombinant enzymes, and cell culture under conditions suitable for the expression of recombinant enzyme replacements. The replacements of recombinant enzymes expressed in the host cell are selected from a group consisting of beta algaidase, laronidase, and recombinant enzyme replacements fused to a therapeutically active polypeptide.

Production of mammalian antibody and antibody fragments In one embodiment of the present invention, production of recombinant single-chain mammalian antibodies, Fab fragments and / or full-chain antibodies or fragments or portions thereof or a host cell of the species P. fluorescens is provided. In one embodiment, after expression of the protein, the protein can be isolated and optionally purified. Optionally, the protein can be renatured to produce an active protein. The antibody or antibody fragments are optionally linked to a secretion signal sequence to be directed to the cells during production. In another modality, a host cell of the species Pseudomonas is provided, which has been transformed to express recombinant antibodies of single-chain mammal, Fab fragments and / or full-chain antibodies or fragments or portions thereof. In one embodiment, the P. fluorescens cell can produce a single chain antibody or fractions or portions thereof. A single chain antibody may include the antigen binding regions of the antibodies on a single, stably folded polypeptide chain. The single chain antibodies are smaller in size than the classical immunoglobulins, but they can retain the specific binding properties of the antigen, of the antibodies. Single-chain antibodies can be used for therapeutic uses, such as "naked" antibodies, "naked" antibodies, bispecific antibody linkers, radioconjugates, or as fusions with effector domains, diagnostic materials, such as tumor imaging, or marker assays. of cancer in vivo or ex vivo, research tools, such as the purification and detection of proteins including the identification and characterization of new therapeutic targets, antibody microarrays, visualization technology and / or vehicles for the distribution of genes or drugs. In yet another embodiment, the P. fluorescens cell produces fragments of Fab or portions thereof. The Fab fragments can be a piece of a particular antiserum. The Fab fragment can contain the antigen binding site. The Fab fragment can contain two chains: a light chain and a heavy chain fragment. These fragments can be linked via a linker or a disulfide bond. In other embodiments of the present invention, full-chain antibodies can be expressed in P. fluorescens, and other species of Pseudomonas. An intact antibody containing the Fe region may be more resistant against degradation and clearance in vivo, thereby having a longer biological half-life in circulation. Such antibodies can be used as a therapeutic agent for diseases that require sustained therapies. In one embodiment, a process for producing a functional antibody or fragment thereof in Pseudomonas is provided by the provision of an expression vector that contains separate cistronic or polycistronic sequences. The separate cistronic expression vector can comprise a first pair of cistron promoter for the expression of an immunoglobulin light chain and a second pair of cistron promoters for the expression of a immunoglobulin heavy chain, such that the Expression of the light chain and the heavy chain are independently regulated by separate promoters. Each cistron within the polynucleotide of the expression cassette may include a translation initiation region (TIR) operably linked to the nucleic acid sequence encoding the light chain or the heavy chain of the full-length antibody. In one embodiment, the TIR sequences can be manipulated to provide different combinations of translational force for light and heavy chains. In an alternative embodiment, a sequence encoding the heavy chain can be located on the same plasmid as a coding sequence of the light chain. In an alternative embodiment, the heavy and light chain sequence are found in a polycistronic sequence within a single plasmid, or encoded within the host genome. In a further embodiment, there is provided a process for producing a functional antibody or fragment thereof in a host cell, and transformed with two separate translational units respectively encoding the light and heavy chains of the antibody. In one embodiment the process includes: a) culturing the host cell under suitable conditions, so that the light chain and the heavy chain are expressed in a sequential manner, whereby the production of the light and heavy chains is temporarily separated and b) allow the assembly of the light and heavy chains to form the functional antibody or fragment thereof. In a further embodiment, the Pseudomonas expression system can express the simple therapeutic human chain, Fab fragments or full-chain antibodies or portions thereof, including, but not limited to, Fab, Fab ', F (ab') 2 , F (ab ') 2- leucine zipper, Fv, dsFv, anti-CD antibody 1 8, chimeric antibodies, human antibodies, humanized antibodies, or those described in table 3 below.

TABLE 3 - Antibodies and antibody fragments Production of Transcriptional Factors In one embodiment of the present invention, the production of mammalian recombinant transcription factors is provided by a host cell of the Pseudomonas fluorescens species. In one embodiment, after expression with the protein, the protein can be isolated. In yet another embodiment, the protein can be purified. Optionally, the protein can be renatured to produce an active protein. In yet another embodiment, a host cell of the Pseudomonas species is provided which has been transformed to express a mammalian recombinant expression factor. Transcription factors, suitable for insertion into the expression systems of the present invention, include those from the spin-helix family and members of the Pac family, as well as other families of transcription factors known in the art. Members of these families, suitable for use with the present invention include homologs and mammalian analogs of: transcription regulators; Transcription factors of the ASNC family such as ASNC-trans-network, putative transcriptional regulators; Bacterial regulatory proteins of family 1 uxR; transcription factors helix-turn-helix bacterial regulators; Bacterial regulatory proteins of the arsR family; transcription factors of the helix domain turn helix, especially the rpiR family; transcription factors of bacterial regulatory proteins; transcription factors helix-turn-helix of bacterial regulators; transcription factors of DNA binding domain; MarR family of transcription factors; ROK family of transcription factors; MerR family of regulatory proteins; arginine repressor transcription factors; transcription factors of firmicute; regulatory transcription factors of iron uptake; sigma transcription factors; transcription factors of the receptor response regulator; transcription factors of the attenuating protein of the binding to the tryptophan RNA; transcription factors of the sugar binding domain-putative; transcription factors of the PRD domain, nitrogen regulatory protein transcription factors; negative regulators of genetic competence, such as MecA; negative transcriptional regulatory transcription factors; bacterial transcripcisinal regulatory transcription factors; transcription factors responsive to glycerol 3-phosphate; iron-dependent repressor transcription factors; and numerous species of specific transcriptional regulatory transcription factors. The transcriptional factors expressed by the Pseudomonas species can be used for diagnostic, therapeutic and research applications.

Preparation of vectors Polynucleotides Mammalian recombinant protein and peptides can be expressed from polynucleotides in which the sequence encoding the target polypeptide is operably linked to the transcriptional and translational regulatory elements that form a functional gene from which the host cell can express the protein. The coding sequence may be a native coding sequence for the target polypeptide, if available, but may also be a coding sequence that has been selected, improved or optimized for use in the selected expression host cell. For example, by synthesis of the gene to reflect the deviation of codon usage of a Pseudomonas species such as P. fluorescens. The gene or genes that result will have been built into or will be inserted into one or more vectors, which will then be transformed into the host cell of reaction. The nucleic acid or a polynucleotide that is said to be provided in an "expressible form" means that the nucleic acid or a polynucleotide that contains at least one gene that can be expressed by the host cell of bacterial expression, selected.

Regulatory elements The regulatory elements used herein may be operably linked to the gene which codes for the target mammalian recombinant protein. The coding sequence of the gene encoding the protein, used herein, may contain, in addition to the sequence coding for the mature polypeptide, and transcriptional regulatory elements, additional coding elements, eg, one or more than the coding sequences for peptide, prepeptide, pro-peptide, pre-pro-peptide, or other commonly used coding elements known in the art, excluding the functional secretion signal peptides in the expression host cell, selected . The term "operably linked (a)" as used herein, refers to any configuration in which the transcriptional or translational regulatory elements are covalently linked to the coding sequence in such or such arrangements, with reference to to the coding sequence, so that the regulatory elements can direct the expression of the coding sequence. In one embodiment, the regulatory elements will be part of a complete gene before undergoing transformation within a host cell; however, in other modalities, the regulatory elements are part of another gene, which may be part of the host's genome, or may be part of a genome of another organism, or may be derived from it.

Promoters and accessory elements Promoters used in accordance with the present invention may be constitutive promoters or regulated promoters. Common examples of useful regulated promoters include those of the family derived from the lac promoter (e.g. the lacZ promoter), especially the tac and tre promoters described in U.S. Patent No. 4,551, 433 to DeBoer, as well as the promoters Ptacld, Ptacl7, and Ptacl l, PlacUVd, and T7lac. Common examples of the non-lac type promoters useful in the expression systems according to the present invention include those listed in Table 4.

TABLE 4 - Examples of non-lac promoters See, for example, J. Sánchez-Romero and V. De Lorenzo (1999) Manual of Industrial Microbiology and Biotechnology (A. Demain and J. Davies, eds.) Pp. 460-74 (ASM Press, Washington, D.C.); H. Schweizer (2001) Current Opinion in Biotechnology 12: 439-445; and R. Slater and R. Williams (2000) Molecular Biology and Biotechnology (J. Walker and R. Rapley, eds.) pp.1 25-54. A promoter having the nucleotide sequence of a native promoter for the bacterial host cell, selected can also be used to control the expression of the transgene encoding a target polypeptide, for example, a Pseudomonas antrinalate or operon promoter. of benzoate (Pant, Pben). Tandem promoters can also be used, in which more than one promoter is covalently linked to another promoter, either in the same or in a different sequence, for example, a Pant-Pben tandem promoter (interpromotor hybrid) or a Tandem promoter Plac-Plac. Regulated promoters utilize regulating promoter proteins in order to control the transcription of the gene of which the promoter is a part. Where a regulated promoter is used herein, a corresponding regulatory promoter protein will also be part of an expression system according to the present invention. Examples of promoter regulatory proteins include: activating proteins, for example, the E. coli catabolite activating protein, MaIT protein; transcriptional activators of the AraC family; repressor proteins, for example, Lad proteins from E. coli; and double-function regulatory proteins, for example, NagC proteins of E. coli. Many regulated promoter pairs / regulatory promoter proteins are known in the art. Promoter regulatory proteins interact with an effector compound, for example, a compound that is reversibly or irreversibly associated with the regulatory protein, to make it possible for the protein to release well bind at least to a regulatory region of the gene's DNA transcript that is under the control of the promoter, with which allows or blocks the action of a transcriptase enzyme at the start of transcription of the gene. The effector compounds are classified as either inducers or co-repressors, and these compounds include native effector compounds and free inductive compounds. Many trios of regulated promoter / promoter regulatory protein / effector compound are known in the art. Also an effector compound can be used throughout cell culture or fermentation, in a mode in which a regulated promoter is used, after the development of a desired quantity or density of the host cell biomass, a effector compound appropriate to the culture, in order to directly or indirectly result in the expression of or of the desired target genes. By way of example, where a promoter of the lac family is used, a lacl gene may also be present in the system. The lacl gene, which is (usually) a constitutively expressed gene, codes for the Lac repressor protein (Lacl protein) that binds to the lac operator of those promoters. Thus, where a promoter of the lac family is used, the lacl gene can also be included and expressed in the expression system. In the case of members of the tac promoter family, for example, the tac promoter, the effector compound is an inducer, such as a free inducer such as I PTG (isopropyl-β-D-1-thiogalactopyranoside, also called "isopropylthiogalactoside").

Other elements Other regulatory elements can be included in an expression construction. Such elements include, but are not limited to, for example, transcription enhancer sequences, translational enhancer sequences, other promoters, activators, translation start and stop signals, transcription terminators, and cistronic regulators, polycistronic regulators. , label sequences, such as the sequences encoding the peptide "markers" and "marker" of the nucleotide sequence, which facilitates the identification, separation, purification or isolation of an expressed polypeptide. To a minimum, a gene encoding a protein according to the present invention may include, in addition to the sequence coding for the mammalian protein, the following regulatory elements operably linked to it: a promoter, a binding site ribosome (RBS), a transcription terminator, start and end signals of the translation. Useful RBSs can be obtained from any of the species useful as host cells in expression systems such as from the selected host cell. Many specific RBSs have a variety of consensus RBSs, are known, for example, those described in and referred to by D. Frishman et al. (1,999) Gene 234 (2): 257-65; and B. E. Suzek et al. (2001) Bioinformatics 17 (12): 1 1 23-30. In addition, either native or synthetic RBSs can be used, for example, those described in: EP 0207459 (Synthetic RBSs); Ikehata et al. (1989) Eur. J. Biochem. 1 81 (3): 563-70 (native RGB sequence of AAGGAAG). Additional examples of translation and transcription methods, vectors and elements, and other elements useful in the present invention are described, for example, in U.S. Patent Nos. 5,055,294 and 5,128,130 to Gilroy et al.; 5,281, 532 to Rammler et al.; 4,695,455 and 4,861, 595 to Barnes et al.; 4,755,465 to Gray et al.; and 5, 1 69,760 to Wilcox.

Vectors The transcription of the DNA encoding the enzymes of the present invention by Pseudomonas is increased by the insertion of an enhancer sequence within the vector or the plasmid. Typical enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in size, which act on the promoter to increase its transcription. Examples include various Pseudomonas enhancers, as described hereinafter. In general, the recombinant expression vectors will include origins of replication and selectable markers that allow the transformation of the host cell Pseudomonas, for example, the antibiotic-free resistance genes of P. fluorescens, and a promoter derived from a highly expressed gene. , to direct the transcription of a downstream structural sequence. Such promoters can be derivatives of the operons that code for enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with the translation initiation and termination sequences and, in one embodiment, a guiding sequence, capable of directing the secretion of the translated enzyme. Optionally, the heterologous sequence can encode a fusion enzyme which includes an N-terminal identification peptide which imparts desired characteristics, for example, stabilization or simplified purification within the expressed recombinant product. Expression vectors useful for use with P. fluorescens in the expression of enzymes, are constructed by inserting a structural DNA sequence encoding a desired protein, together with the appropriate translation initiation and termination signals., in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication, to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Vectors are known in the art as useful for the expression of recombinant proteins in host cells, and any of these can be used to express the genes according to the present invention. Such vectors include, for example, plasmids, cosmids, and phage display vectors. Examples of useful plasmid vectors, include, but are not limited to, expression plasmids pBBRIMCS, pDSK519, pKT240, pML122, pPS10, RK2, RK6, pRO1600, and RSF1010. Other examples of such useful vectors include those described for example by N. Hayase (1994) Appl. Envir. Microbiol. 60 (9): 3336-42; A. A. Lushnikov et al. (1985) Basic Life Sci. 30: 657-62; S. Graupner and W. Wackernagel (2000) Biomolec. Eng. 17 (1): 11-16; H. P. Schweizer (2001) Curr. Opin. Biotech 12 (5): 439-45; M. Bagdasarian and K. N. Timmis (1982) Curr. Microbiol. Immunol 96: 47-67; T. Ishii et al. (1994) FEMS Microbiol. Lett 116 (3): 307-13; I. N. Olekhnovich and Y. K. Fomichev (1994) Gene 140 (1): 63-65; M. Tsuda and T. Nakazawa (1993) Gene 136 (1-2): 257-62; C. Nieto et al (1990) Gene 87: 145-49; J. D. Jones and N. Gutterson (1987) Gene 61 (3): 299-306; M. Bagdasarian et al. (1981) Gene 16 (1-3): 237-47; H. P. Schweizer et al. (2001) Genet. Eng. (NY) 23: 69-81; P. Mukhopadhyay et al. (1990) J. Bact. 172: 477-80; D. O.

Wood et al. (1981) J. Bact. 145 (3): 1448-51; and R. Holtwick et al. (2001) Microbiology 147 (Pt 2): 337-44. Additional examples of expression vectors that may be useful in Pseudomonas host cells include those listed in Table 5 as they are derived from the indicated replicons.

TABLE 5 - Some examples of useful expression vectors The expression plasmid, RSF1010, is described, for example, by F. Heffron et al. (1975) Proc. Nat'l Acad. Sci. USA. 72 (9): 3623-27, and by K. Nagahari & K. Sakaguchi (1 978) J. Bact. 133 (3): 1 527-29. Plasmid RSF1010 and derivatives thereof are particularly useful vectors in the present invention. Useful derivatives of RSF1 01 or specimens, which are known in the art, include for example, pKT21 2, pKT214, pKT231 and related plasmids, and pMYC1 050 and related plasmids (see for example, U.S. Pat. Nos. 5 , 527883, and 5,840,554 to Thomson et al.), Such as, for example, pMYC1803. Plastic pMYC1 803 is derived from plasmid pTJS260 based on RFS 1010 (US Patent No. 5,169,760 to Wilcox), which carries a regulated tetracycline resistance marker and the replication and mobilization loci from the plasmid RSF 1 01. Other useful, exemplary vectors include those described in U.S. Patent No. 4,680,264 to Puhier et al. In one embodiment, an expression plasmid is used as the expression vector. In yet another embodiment, RSF 101 0 or a derivative thereof is used as the expression vector. In still another embodiment, pMYC1 050 or a derivative thereof, or a pMYC1803 or a derivative thereof, is used as the expression vector. The plasmid can be maintained in the host cell by the use of a selection marker gene, also present in the plasmid. This may be an antibiotic resistance gene or genes in which case the corresponding antibiotic or antibiotics will be added to the fermentation medium, or any other type of selection marker gene known as useful in the art, for example, a prototrophic restoration gene. in which case the plasmid will be used in a host cell is auxotrophic for the corresponding trait, for example, a biocatalytic trait or characteristic such as a biosynthesis of amino acid or a trait of nucleotide biosynthesis or a utilization trait of carbon source. Extensive sequence information required for molecular genetics and genetics techniques is widely publicly available. Access to complete mammalian nucleotide sequences, as well as human sequences, genes, cDNA sequences, amino acid sequences and genomes, can be obtained from GenBank at the URL address: http: // www. ncbi.nlm. nih .gov / Entrez. Additional information can also be obtained from GeneCards, an electronic encyclopedia that integrates information about genes and their products and the biomedical applications of the Weízmann Institute of Genome Science and Bioinformatics (http://bioinformatics.weizmann.ac.il/cards/), the nucleotide sequence information can also be obtained from the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/ embl /) or DNA or Japan Data Bank (DDBJ, http: //www.ddbj.mig.ac.jp/; Additional sites for information on amino acid sequences include the information resource network site of protein from Georgetown (http: // www. nbrf.georgetown.edu/pir/) and Swiss-Prot (http: //au.expasy.org/ sprot / sprot-top. htmi).

Transformation Transformation of Pseudomonas host cells with the vector (s) can be performed using any transformation methodology known in the art, and bacterial host cells can be transformed as intact cells or as protoplasts (eg, including cytoplasts). Exemplary transformation methodologies include poration methods, for example, electroporation, protoplast fusion, bacterial conjugation, and treatment with divalent cations, for example, calcium chloride treatment or CaCl / Mg2 + treatment, or other well-known methods in The technique. See for example, Morrison (1 977) J. Bact. 132: 349-351; Clark-Curtiss and Curtiss (1983) Methods in Enzymology 101: 347-362, Sambrook et al. (1 989) Molecular Cloning, A Laboratory Manual (2nd ed.); Kriegler (1990) Gene Transfer and Expression: A Laboratory Manual; and Ausubel et al. , eds. (1 994) Current Protocols in Molecular Biology.

Pseudomonas Organisms While the first invention herein is the use of Pseudomonas fluorescens, other organisms of Pseudomonas and closely related bacteria may be useful. The Pseudomonas and closely related bacteria are generally part of the group defined as "Protobacteria Gram (-) Subgroup 1" or "Gram-negative aerobic bacilli or cocci" (Buchanan and Gibbons (eds.) (1 974) Bergey's Manual of Determinative Bacteriology , pp. 217-289).

TABLE 6. "Gram-negative aerobic bacilli or cocci" (Bergey (1974)) The "Protobacteria Gram (-) Subgroup 1" also include proteobacteria that can be classified in this heading according to the criteria used in the classification. The heading also includes groups that were previously classified in this section but no longer, such as the genera Acidovorax, Brevundimonas, Burkholderia, Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, the genus Sphingomonas (and the genus Blastomonas, derived from it), which was created by the regrouping of organisms belonging to (and previously called, species of the genus Xanthomonas, the genus Acidomonas, which was created by the regrouping of organisms belonging to the genus Acetobacter as defined in Bergey (1 974). In addition, hosts may include cells of the genus Pseudomonas, Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciens (ATCC 19375), and Pseudomonas putrefaciens (ATCC 8071), which have been respectively reclassified as Alteromonas haloplanktis, Alteromonas nigrifaciens and Alteromonas putrefaciens. , for example, Pseudomonas acidovorans (ATCC 1 5668) and Pseudomonas testosteroni ( ATCC 1 1 996) that have been reclassified as Comamonas acidovorans and Comamonas testosteroni ^ respectively; and Pseudomonas nigrifaciens (ATCC 1 9375) and Pseudomonas piscicida (ATCC 15057) have respectively been reclassified as Pseudomonas nigrifaciens and Pseudoalteromonas piscicida. "Gram-negative proteobacteria subgroup I" also include protobacteria classified as belonging to any of the family: Pseudomonadaceae, Azotobacteraceae (also frequently referred to by the synonym, "azotobacter group" of Pseudomonadaceae), Rhizobiaceae, and Methylomonadaceae (now frequently called by the synonym "" Mef / iy / coccaceae "). Consequently, in addition to those genera otherwise described herein, the additional protobacterial genera that fall within" Subgroup 1 of Gram-negative proteobacteria "include: 1) bacteria of the group Azotobacter of the genus Azorhizophilus, 2) bacteria of the family Pseudomonadaceae of the genus Cellvibrio, Oligella, and Teredinibacter, 3) bacteria of the Rhizobiaceae family of the genera Chelatobacter, Ensifer, Liberibacter (also called "Candidatus Liberibacter"), and Sinorhizobium; 4) Bacteria of the Methylococcaceae family of the genera Methylobacter, Methylocaldum, Methylomic robium, Methylosarcina, and Methylosphaera. In yet another embodiment, the host cell is selected from the "Gram-negative proteobacteria subgroup 2". "Gram-negative proteobacteria subgroup 2" is defined as the group of proteobacteria of the following genera (with the total number of strains of the same deposited, publicly available, listed in the catalog, indicated in parentheses, all deposited in the ATCC, except as is indicated otherwise): Acidomonas (2); Acetobacter (93); Gluconobacter (37); Brevundimonas (23); Beijerinckia (1 3); Derxia (2); Brucella (4); Agrobacterium (79); Chelatobacter (2); Ensifer (3); Rhizobium (144); Sinorhizobium (24); Blastomonas (1); Sphingomonas (27); alcaligenes (98); Bordetella (43); Burkholderia (73); Ralstonia (33) Acidovorax (20); Hydrogenophaga (9); Zoogloea (9); Methylobacter (2) Methylocaldum (1 in NCI MB); Methylococcus (2); Methylomicrobium (2) Methylomonas (9); Methylosarcin (1); Methylosphaera; Azomonas (9); Azorhizophilus (5); Azotobacter (64); Cellvibrio (3); Oligella (5); Pseudomonas (1 139); Francisella (4); Xanthomonas (229); Stenotrophomonas (50); and Oceanimonas (4). The exemplary host cell species of "Gram-negative proteobacteria subgroup 2" include but are not limited to the following bacteria (with the ATCC or other deposit numbers of the secondary strains thereof shown in parentheses): Acidomonas methanolica (ATCC 43581); Acetobacter aceti (ATCC 15973); Gluconobacter oxydans (ATCC 1 9357); Brevundimonas diminuta (ATCC 1 1568); Beijerinckia indica (ATCC 9039 and ATCC 1 9361); Derxia gummosa (ATCC 15994); Brucilla melitensis (ATCC 23456), Brucella abortus (ATCC 23448); Agrobacterium tumefaciens (ATCC 23308), Agrobacterium radiobacter (ATCC 1 9358), Agrobacterium rhizogenes (ATCC 1 1 325); Chelatobacter heintzií (ATCC 29600); Ensifer adhaerens (ATCC 33212); Rhizobium leguminosarum (ATCC 1 0004); Sinorhizobium fredii (ATCC 35423); Blastomonas notatoria (ATCC 35951); Sphingomonas paucimobilis (ATCC 29837); Alcaligenes faecalis (ATCC 8750); Bordetella pertussis (ATCC 9797); Burkholderia cepacia (ATCC 2541 6); Ralstonia pickettii (ATCC 2751 1); Acidovorax facilis (ATCC 1 1228); Hydrogenophaga flava (ATCC 33667); Zoogloea ramigera (ATCC 1 9544); Methylobacter luteus (ATCC 49878); Methylocaldum gracile (NCI MB 1 1 912); Methylococcus capsulatus (ATCC 1 9069); Methylomicrobium agitate (ATCC 35068); Methylomonas methanica (ATCC 35067); Methylosarcina fibrata (ATCC 700909); Methylosphaera hansonii (ACAM 549); Azomonas agilis (ATCC 7494); Azorhizophilus paspali (ATCC 23833); Azotobacter chroococcum (ATCC 9043); Cellvibrio mixtus (UQM 2601); Oligella urethralis (ATCC 1 7960); Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC 35858); Francisella tularensis (ATCC 6223); Stenotrophomonas maltophilia (ATCC 13637); Xanthomonas campestres (ATCC 3391 3); and Oceanimonas doudoroffii (ATCC 27123). In another embodiment, the host cell is selected from "Gram-negative proteobacteria subgroup 3". "Gram-negative proteobacteria subgroup 3" is identified as the group-of proteobacteria of the following genus: Brevundimonas; Agrobacterium; Rhizobium; Sinorhizobium; Blastomonas; Sphingomonas; Alcaligenes; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter; Metylocaldum; Methylococcus; Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus; Azobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacer; Francisella; Stenotrophomonas; Xanthomonas; and Oceanimonas. In yet another embodiment, the host cell is selected from "Gram-negative proteobacteria subgroup 4". "Gram-negative proteobacteria subgroup 4" is defined as the group of protobacteria of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium; Methylomas;; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas; and Oceanimonas. In one embodiment, the host cell is selected from "Gram-negative proteobacteria subgroup 5". "Gram-negative proteobacteria subgroup 5" is defined as the group of protobacteria of the following genera: Methylobacter; Methylocaldum; Methylococcus; M thylomicrobium; Methylomonas; Methylosarcina; Methylophaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Fransicella; Stenotrophomonas; Xanthomonas; and Oceanimonas. The host cell is selected from "Gram-negative proteobacteria subgroup 6". "Gram-negative proteobacteria subgroup 6" is defined as the group of protobacteria of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Stenotrophomonas; Xanthomonas; and Oceanimonas. The host cell is selected from "Gram-negative proteobacteria subgroup 7". "Gram-negative proteobacteria subgroup 7" is defined as the group of protobacteria of the following genera: Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter; Stenotrophomonas; Xanthomonas; and Oceanimonas. The host cell is selected from "Gram-negative proteobacteria subgroup 8". "Gram-negative proteobacteria subgroup 8" is defined as the group of protobacterlas of the following genera: Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas; Stenotrophomonas; Xanthomonas; and Oceanimonas. The host cell is selected from "Gram-negative proteobacteria subgroup 9". "Gram-negative proteobacteria subgroup 9"is defined as the group of protobacteria of the following genera: Brevundimonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas; Stenotrophomonas; and Oceanimonas. The host cell is selected from "gram-negative proteobacteria subgroup 10". "Gram-negative proteobacteria subgroup 10" is defined as the group of protobacteria of the following genera: Burkholderia; Ralstonia; Pseudomonas; Stenotrophomonas; and Xanthomonas. The host cell is selected from "gram-negative proteobacteria subgroup 1 1". "Gram-negative proteobacteria subgroup 1 1" is defined as the group of protobacteria of the following genera: Pseudomonas; Stenotrophomonas; and Xanthomonas. The host cell is selected from "gram-negative proteobacteria subgroup 1 2". "Gram-negative proteobacteria subgroup 1 2" is defined as the group of protobacteria of the following genera: Burkholderia; Ralstonia; Pseudomonas. The host cell is selected from "gram-negative proteobacteria subgroup 1 3". "Gram-negative proteobacteria subgroup 1 3" is defined as the group of protobacteria of the following genera: Burkholderia; Ralstonia; Pseudomonas; and Xanthomonas. The host cell is selected from "gram-negative proteobacteria subgroup 14". "Gram-negative proteobacteria subgroup 14" is defined as the group of protobacteria of the following genera: Pseudomonas and Xanthomonas. The host cell is selected from "gram-negative proteobacteria subgroup 15". "Gram-negative proteobacteria subgroup 1 5" is defined as the group of protobacteria of the genus Pseudomonas. The host cell is selected from "gram-negative proteobacteria subgroup 1 6". "Gram-negative proteobacteria subgroup 1 6" is defined as the group of protobacteria of the following Pseudomonas species (with the ATCC deposit numbers or other deposits of exemplary strains shown in parentheses) P. abietaniphila (ATCC 700689); P. aeruginosa (ATCC 10145); P. alcaligenes (ATCC 14909); P. anguiiliseptica (ATCC 33660); P. citronellolis (ATCC 1 3674); P. flavescens (ATCC 51555); P. mendocin (ATCC 2541 1); P. nitroreducens (ATCC 33634); P. oleovorans (ATCC 8062); P. pseudoalcaligenes (ATCC 17440); P. resinovorans (ATCC 14235); P. straminea (ATCC 33636); P. agarici (ATCC 25941); P. alcaliphila; P. alginovora; P. andersoníi; P. asplenii (ATCC 23835); P. azelaica (ATCC 271 62); P. beijerinckii (ATCC 1 9372); P. borealis; P. boreopolis (ATCC 33662); P. brassicacearum; P. butanovora (ATCC 43655); P. cellulosa (ATCC 55703); P. aurantiaca (ATCC 33663); P. chlororaphis (ATCC 9446, ATCC 1 3985, ATCC 17418, ATCC 17461); P. frag / (ATCC 4973); P. lundensis (ATCC 49968); P. taetrolens (ATCC 4683); P. cissicola (ATCC 3361 6); P. coronafaciens; P. diterpeniphila; P. elongata (ATCC 1 0144); P. flectens (ATCC 1 2775); P. azotoformans; P. benneri, P. cedrella; P. corrúgala (ATCC 29736); P. extremorientalis; P. fluorescens (ATCC 35858); P. gessardii; P. libanensis; P. mandelii (ATCC 700871); P. marginalis (ATCC 10844); P. migulae; P. mucidolens (ATCC 4685); P. orientalis; P. rhodesiae; P. synxantha (ATCC 9890); P. tolaasii (ATCC 3361 8); P. veronii (ATCC 700474); P. frederiksbergensis; P. geniculata (ATCC 1 9374); P. gingeri; P. graminis; P. grimontii; P. halodenitrificans; P. halophila; P. hibiscicola (ATCC 1 9867); P. huttiensis (ATCC 14670); P. hydrogenovora; P. jessenii (ATCC 700870); P. kilonesis; P. lanceolata (ATCC 14669); P. // * / 7 /; P. marginata (ATCC 25417); P. mephitica (ATCC 33665); P. denitrificans (ATCC 1 9244); P. pertucinogen (ATCC 1 90); P. pictorum (ATCC 23328); P. psychorophila; P. fulva (ATCC 3141 8); P. monteilii (ATCC 700476); P. mosselii; P. oryzihabitans (ATCC 43272); P. plecoglossicide (ATCC 700383); P. puf / da (ATCC 12633); P. reactans; P. spinosa (ATCC 14606);; P. baleárica; P. tuteóla (ATCC 43273); P. stutzeri (ATCC 1 7588); P. amygdali (ATCC 33614); P. avellanae (ATCC 700331); P. caricapapayae (ATCC 3361 5); P. cichorti (ATCC 10857); P. ficuserectae (ATCC 35104); P. fuscovag / nae; P. meliae (ATCC 33050); P. syringae (ATCC 1 931 0); P. viridiflava (ATCC 1 3223); P. thermocarboxidovorans (ATCC 35961); P. thermotolerans; P. thivervalensis; P. vancouverensis (ATCC 700688); P. wisconsinensis; Y P. xiamenensis. The host cell is selected from "gram-negative proteobacteria subgroup 17". "Gram-negative proteobacteria subgroup 17" is defined as the group of protobacteria known in the art as "fluorescent pseudomonades" which include those which belong, for example, to the following species of Pseudomonas: P. azotoformans; P. brenneri; P. cedrella; P. corrugata; P. extremorientalis; Pseudomonas fluorescens; P. gessardii; P. libanensis; Pseudomonas mandelii; P. marginalis; P. migulae; P. mucidolens; P. orientalis; P. rhodesiae; P. synxantha; P. tolaasti; and P. veronii. The host cell can be selected from "gram-negative proteobacteria subgroup 18". "Gram-negative proteobacteria subgroup 18" is defined as the group of all subspecies, strains, strains and other sub-special units of the species P. fluorescens, including those belonging, for example, to the following (with the numbers of deposit in ATCC or other numbers of exemplary strains shown in parentheses): P. fluorescens biotype A, also called biovar 1 or biovar I (ATCC 1 3525); P. fluorescens biotype B, also called biovar 2 or biovar I I (ATCC 1 781 6); P. fluorescens biotype C, also called biovar 3 or biovar l l l (ATCC 17400); P. fluorescens biotype F, also called biovar 4 or biovar IV (ATCC 12983); P. fluorescens biotype G, also called biovar 5 or biovar V (ATCC 1751 8); P. fluorescens biovar VI; P. fluorescens Pf0-1; P. fluorescens Pf-5 (ATCC BAA-477); P. fluorescens SBW25; and P. fluorescens Subs .. cellulose (NCI MB 1 0462). The host cell can be selected from "gram-negative proteobacteria subgroup 1 9". "Gram-negative proteobacteria subgroup 19" is defined as the group of all strains of P. fluorescens biotype A. A particular strain of this biotype is P. fluorescens strain MB101 (see U.S. Patent No. 5,169,760 to Wilcox), and derived from them. An example of a derivative thereof is P. fluorescens strain MB214, constructed by inserting into the chromosomal locus of MB101 asd (aspartate dehydrogenase gene), a native construct of E. coli PlacM-lacl-lacZYA (e.g. which PlacZ was deleted). In one embodiment, the host cell is any of the Pseudomonadales Proteobacteria. In a particular embodiment, the host cell is any of the Proteobacteria of the psedomonadaceae family. Additional P. fluorescens strains that can be used in the present invention include P. fluorescens Migula and P. fluorescens Loitokitok, which have the following designations from the ATCC: (NCIB 8286); NRRL B-1244; strain NCIB 8865 COI; strain NCIB 8866, strain C02; 1291 (ATCC 17458; IFO 15837; NCIB 8917; LA; NRRL B-1864; pyrrolidine; PW2 (ICMP 3966; NCPPB 967; NRRL B-899); 13475; NCTC 10038; NRRL B-1603 (6; IFO 15840); 52-1C; CCEB 488A (BU 140); CCEB 553 (IEM 15/47); IAM 1008; (AHH-27); IAM 1055 (AHH-23); 1 (IFO 15842); 12 (ATCC 25323; NIH 11; den Dooren de Jong 216); 18 (IFO 15833; WRRL P-7); 93 (TR-10); 108 (52-22; IFO 15832); 143 (IFO 15836; PL); 149 (2-40-); 40; IFO 15838); 182 (IFO 3081; PJ 73); 184 (IFO 15830); 185 (W2 L-1); 186 (IFO 15829; PJ 79); 187 (NCPPB 263); 188 (NCPPB 316); 189 (PJ227; 1208); 191 (IFO 15834; PJ 236; 22/1); 194 (Klinge R-60; PJ 253); 196 (PJ288); 197 (PJ 290); 198 (PJ 302); PJ 368), 202 (PJ 372), 203 (PJ 376), 204 (IFO 15835, PJ 682), 205 (PJ 686), 206 (PJ 692), 207 (PJ 693), 208 (PJ 722), 212 (PJ 832); 215 (PJ 849); 216 (PJ 885); 267 (B-9); 271 (B-1612); 401 (C71A; IFO 15831; PJ 187); NRRL B-3178 (4; IFO 15841); KY 8521; 3081; 30-21; (IFO 3081; N; PYR; PW; D946-B83 (BU 2183; FERM-P 3328); P-2563 (FERM-P 2894; IFO 13658); IAM-1126 (43F); M-1; A506 (A5-06); A505 (A5-05-1); A526 (A5-26); B69; 72; NRRL B-4290; PMW6 (NCIB 11615); SC 12936; A1 (IFO 15839); F 1847 (CDC-EB); F 1848 (CDC 93); NCIB 10586; P17; F-12; AmMS 257; PRA25; 6133D02; 6519E01; N1; SC15208; BNL-WVC; NCTC 2853 (NCIB 8194); H13; 1013 (ATCC 11251; CCEB 295); IFO 3903; 1062; or Pf-5.

Fermentation The term "fermentation" includes both modalities in which literal fermentation is employed, and modalities in which other modes of non-fermentative cultivation are employed. Fermentation can be done at any scale. In one embodiment, the fermentation medium can be selected from rich media, minimal media, and mineral salt media; A rich medium can be used, but it is typically avoided. In yet another embodiment, either a minimal medium or a mineral salt medium is selected. In yet another mode, a minimum means is selected. The mineral salt medium consists of mineral salts and a carbon source such as, for example, glucose, sucrose or glycerol. Examples of mineral salt media include, for example, M9 medium, Pseudomonas medium (ATCC 179), Davis medium and Mingioli (see, BD Davis and ES Mingioli (1 950) J. Bact. 60: 17-28 ). The mineral salts used to make the mineral salt media include those selected from, for example, potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates. of iron, manganese and zinc. Typically, no source of organic nitrogen, such as peptone, tryptone, amino acids, or a yeast extract, in a mineral salt medium is included. Rather, an inorganic nitrogen source is used, and this can be selected from, for example, the ammonium salts, aqueous ammonia and gaseous ammonia. A mineral salt medium will typically contain glucose as a carbon source. In comparison to the mineral salt media, the minimum media may also contain mineral salts and a carbon source, but can be supplemented with, for example, low levels of amino acids, vitamins, peptones, or other ingredients, although these are added at very minimal levels. In one embodiment, the medium can be prepared using the components listed in the following table 7. The components can be added in the following order: firstly (NH4) H P04, KH2P04 and citric acid can be dissolved in approximately 30 liters of distilled water, then a solution of trace elements can be added, followed by the addition of a antifoaming agent, such as Ucolub N 1 1 5. Then, after heat sterilization (such as at about 121 ° C), sterile solutions of glucose, magnesium sulfate and thiamine hydrochloride can be added. The pH control approximately 6.8 can be achieved using aqueous ammonia. Sterile distilled water can then be added to adjust the initial volume to 371 less than the glyceroi stock (12 ml). Chemical products are commercially available from several suppliers, such as Merck. This medium can allow a high cell density culture (HCDC) for the development of species and Pseudomonas and related bacteria. The HCDC can start with a batch process that is followed by a two-phase feed batch culture. After unlimited growth in the batch part, the growth can be controlled at a reduced specific growth rate, in a period of three times of duplication in which the concentration of the biomass can be increased several times. Additional details of the culture procedures are reported by Riesenberg, D et al. (1991) "High cell density cultivation of Escherichia coli at controlled specific growth rate" J Biotechnol 20: 17-27.

Table 7: Composition of the medium Component Initial concentration KH2P04 13.3 g I'1 (NH4) 2HP04 4.0 g I "1 Citric acid 1 .7 g 1" 1 MgSO4-7H20 1.2 g I "1 Trace metal solution 10 ml AND thiamine HCl 4.5 mg 1 ~ 1 Glucose-H20 27.3 g 1"1 Antifoam Ucolub N115 0.1 ml 1" 1 Feeding solution MgS04-7H20 19.7 g Y Glucose-H20 770 g ° "1 NH3 23 g Trace metal solution Fe cit (lll) 6 g and MnCI2-4H20 1.5 g 1"1 ZmCH2COOL2-2H20 0.8 and H3B03 0.31" 1 Na2Mo04-2H20 0.251"1 CoCI26H20 0.25 and CuCI22H20 0.15 r1 Ethylene 0.84 and Dinitrile-tetraacetic acid Na2-2H20 (Tritriplex III, Merck) The expression system according to the present invention can be cultured in any fermentation format. For example, fermentation modes batch fed, semi-continuous and continuous can be employed herein. The expression systems according to the present invention are useful for the expression of transgenes at any scale (eg, volume) of fermentation. Thus, for example, fermentation volumes can be used at the microliter scale, at the scale of centriliter or at the deciliter scale; and fermentation volumes at 1 liter scale and larger can be used. In one embodiment, the volume of fermentation will be or above 1 liter. In a further mode, the volume of fermentation will be at or above 5 liters, 10 liters, 15 liters, 20 liters, 25 liters, 50 liters, 75 liters, 1 00 liters, 200 liters, 500 liters, 1, 000 liters , 2,000 liters, 5,000 liters, or 50,000 liters. In the present invention, the growth, cultivation, and / or fermentation of the transformed host cells is performed within a temperature range that allows the survival of the host cells, such as a temperature within the range of about 4 ° C. at about 55 ° C, inclusive. In addition, "growth" is used to indicate biological states and / or enlargement, as well as biological results in which a cell that is not divided and / or enlarged is being metabolically supported, making it synonymous with last use with the term "maintenance" " Cellular density An additional advantage in the use of P. fluorescens in the expression of mammalian recombinant proteins, includes the ability of P. fluorescens to be developed at high cell densities in comparison to £. coli or other bacterial expression systems. For this purpose, the expression systems of P. fluorescens according to the present invention can provide a cell density of approximately 20 g / liter or more. The expression systems of P. fluorescens according to the present invention can likewise provide a cell density of at least about 70 g / liter, as stated in terms of biomass by volume, the biomass being measured as the dry cell weight . In one embodiment, the cell density will be at least 20 g / liter. In yet another embodiment, the cell density will be at least 25 g / liter, 30 g / liter, 35 g / liter, 40 g / liter, 45 g / liter, 50 g / liter, 60 g / liter, 70 g / liter. liter, 80 g / liter, 90 g / L, 1 00 g / liter, 1 1 0 g / liter, 1 20 g / liter, 1 30 g / liter, 140 g / liter, or at least 1 50 g / L. In other modalities, the cell density in the induction will be between 20 g / liter, and 150 g / L; , 20 g / liter, and 120 g / L; 20 g / liter, and 80 g / L; 25 g / liter, and 80 g / L; 30 g / liter, and 80 g / liter, 35 g / liter, and 80 g / liter, 40 and 80 g / liter, 45 g / liter, and 80 g / liter, 50 g / liter, and 75 g / l; 50 g / liter, and 70 g / L; 40 g / liter, and 80 g / L.

Isolation and Purification The proteins of this invention can be further purified to substantial purity by standard techniques well known in the art, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anionic and cation exchange chromatography. , phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, nickel chromatography, hydroxylapatite chromatography, reverse phase chromatography, lectin chromatography, preparative electrophoresis, solubilization with detergent, selective precipitation with substances such as column chromatography, immunopurification and others. For example, proteins that have established molecular adhesion properties can be reversibly fused to a ligand. With the appropriate ligand, the protein can be selectively adsorbed to a purification column and then fed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. In addition, the protein can be purified using immunoaffinity columns or ni-NTA columns. The general techniques are also described in, for example, R. Scopes (1982) Protein Purification: Principles and Practice, Springer-Verlag: N. Y.; Deutscher (1990) Guide to Protein Purification, Academic Press; U.S. Patent No. 4,51 1, 503; S. Roe (2001) Protein Purification Techniques: A Practical Approach, Oxford Press; D. Bollag, et al. (1 996) Protein Methods, Wiley-Lisa, Inc.; AK Patra et al. (2000) Protein Expr Purif, 1 8 (2): 1 82-92; and R. Mukhija, et al. (1 995) Gene 165 (2): 303-6. See also, for example, Deutscher (1990) "Guide to Protein Purification", Methods in Enzymology vol. 1 82, and other volumes in this series; Coligan, et al. (1996 and periodic supplements) Current Protocols in Protein Science, Wiley / Greene, NY; and the literature of the manufacture on the use of products for protein purification, for example, Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. The combination with recombinant techniques allows fusion to appropriate segments, for example, to a FLAG sequence in an equivalent that can be fused via a protease-removable sequence. See also, for example: Hochuli (1989) Chemische Industrie 12: 69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principie and Methods 12: 87-98, Plenum Press, NY; and Crowe, et al. (1992): QIAexpresss: The High Level Expression & Protein Purification System QU IAGEN, Inc., Chatsworth, Calif. The detection of the expression protein is achieved by methods known in the art and includes, for example, radioimmunoassays, Western blotting technique or immunoprecipitation. The recombinantly produced and expressed enzyme can be recovered and purified from the recombinant cell cultures by various methods, for example, high performance liquid chromatography (HPLC), can be employed for the final purification steps, as necessary. Certain proteins expressed in this invention can form insoluble aggregates ("inclusion bodies"). Various protocols for the purification of proteins from inclusion bodies are suitable. For example, purification of inclusion bodies typically involves extraction, separation and / or purification of inclusion bodies by disintegrating the host cells, for example, by incubation in a 50 mM Tris / HCl buffer, pH 7.5, chloride 50 mM sodium, 5 mM magnesium chloride, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension is typically used using 2-3 passes through a French press. The cell suspension can also be homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternative methods for lysis of bacteria are apparent to those skilled in the art (see, for example, Sambrook, J., EF Fritsch and T. Maniatis eds. (1989) "Molecular Cloning: A Laboratory Manual", 2nd ed. , Cold Spring Harbor Laboratory Press, Ausubel et al., Eds. (1 994) Current Protocols in Molecular Biology). If necessary, the inclusion bodies can be solubilized, and the lysed cell suspension can typically be centrifuged to remove undesirable insoluble matter. The proteins that formed the inclusion bodies can be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4M to about 8M), formamide (at least about 80%, volume / volume basis), and guanidine hydrochloride (from about 4M to about 8M ). Although guanidine hydrochloride and similar agents are denaturing, this denaturation is not irreversible and renaturation can occur after elimination (by dialysis, for example) or dilution of the denaturant, allowing the re-formation of the protein immunologically and / or biologically active Other suitable shock absorbers are known to those skilled in the art. Alternatively, it is possible to purify the recombinant proteins from the periplasm of the host. After lysis of the host cell, when the recombinant protein is exported to the periplasm of the host cell, the periplasmic fraction of the bacterium can be isolated by cold osmotic shock in addition to other methods known to those skilled in the art. To isolate the recombinant proteins from the periplasm, for example, the bacterial cells can be centrifuged to form a pellet. The pellet can be resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria can be centrifuged and the pellet can be resuspended in ice-cold 5 mM magnesium sulfate and kept in an ice bath for approximately 10 minutes. The cell suspension can be centrifuged and the supernatant decanted and stored. The recombinant proteins present in the supernatant can be separated from the host proteins by standard techniques and repair well known to those skilled in the art. An initial salt fractionation can separate many of the unwanted proteins from the host cell (or proteins derived from the cell culture medium) from the recombinant protein of interest. Such an example can be ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. The proteins are then precipitated based on their solubility. The more hydrophobic a protein is, the more likely it is to precipitate it at lower concentrations of ammonium sulfate. A typical protocol includes the addition of saturated ammonium sulfate to a protein solution, so that the resulting concentration of ammonium sulfate is between 20-30%. This concentration will precipitate the most hydrophobic proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant at a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt is removed if necessary, either through dialysis or diafiltration. Other methods that rely on the solubility of proteins such as ethanol precipitation are well known to those skilled in the art, and can be used to fractionate whole protein mixtures. The molecular weight of a recombinant protein can be used to isolate it from larger or smaller proteins, using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture can be ultrafiltered through a membrane with a pore size having a lower molecular weight cutoff than the molecular weight of the protein of interest. The retentate of the ultrafiltration can then be ultrafiltered against a membrane with a molecular cut greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane to the filtrate. The filtrate can then be subjected to chromatography as described below. The recombinant proteins can also be separated from other proteins based on their size, their net surface charge, hydrophobicity, and affinity for the ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and immunoprecipitated proteins. All these methods are well known in the art It will be apparent to a skilled person that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (for example, Pharmacia Biotech).

Renaturing and Refolding The insoluble protein can be renatured or refolded to generate the secondary or tertiary configuration of the protein structure. Protein refolding steps may be used as necessary to complete the configuration of the recombinant product. Refolding and renaturation can be accomplished by using an agent that is known in the art to promote the dissociation / association of proteins. For example, the protein can be incubated with dithiothreitol followed by incubation with the disodium salt of the oxidized glutathione followed by incubation with a buffer containing a refolding agent such as urea. The recombinant protein can also be renatured, for example, by dialyzing it against phosphate buffered saline (PBS) or 50 mM sodium acetate, buffer pH 6 plus 200 mM sodium chloride. Alternatively, the protein can be refolded while immobilized on a column, such as the Ni-NTA column by the use of a linear gradient of 6M-1 M urea in 500 mM sodium chloride, 20% glycerol, Tris / HCl 20 mM, pH 7.4, containing protease inhibitors. The renaturation can be in a period of 1.5 hours or more. After renaturation, the proteins can be eluted by the addition of 250 mM imidazole. The imidazole can be removed by a final dialysis step against PBS or 50 mM sodium acetate buffer, pH 6 plus 200 mM sodium chloride. The purified protein can be stored at 4 ° C or frozen at -80 ° C. Other methods include, for example, those that can be described in: MH Lee et al. (2002) Protein Expr. Purif 25 (1): 1 66-73; W. K. Cho et al. (2000) J Biotechnology 77 (2-3): 169-78; Deutscher (1990) "Guide to Protein Purification", Methods in Enzymology vol.1 82, and other volumes in these series; Coligan, et al. (1 996 and periodic supplements) Current Protocols in Protein Science, Wiley / Greene, NY; S. Roe (2001) Protein Purification Techniques: A Practical Approach, Oxford Press; D. Bollag, et al. (1 996) Protein Methods, Wiley-Lisa, Inc.

Peptide or active protein analysis Typically, an "active" protein includes proteins that have a biological function or biological effect comparable to the corresponding native protein. In the context of proteins, this typically means that a polynucleotide or polypeptide comprises a biological function or effect having at least about 20%, about 50%, about 60%, about 70%, about 75%, about 80% , approximately 85%, approximately 90%, approximately 95%, approximately 98% or 100% of biological function in comparison to the corresponding native protein using standard parameters. The determination of the activity of the protein can be carried out using comparative, directed, standard, biological assays for particular proteins. An indication that a biological function of recombinant protein or effect thereof is that the recombinant polypeptide is immunologically cross-reactive with the native polypeptide. The active proteins typically have a specific activity of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of that of the native mammalian protein. In addition, the substrate specificity (kcat / Km) is optionally substantially similar to the native mammalian protein. Typically, kcat / Km will be at least 30%, 40%, 50%, 60%, 70%, or 90% that of the native protein. Methods for evaluating and quantifying protein measurements and peptide activity and substrate specificity (kcat / Km) are well known to those skilled in the art.

The activity of a mammalian recombinant protein can be measured by any conventional or standard in vitro or in vitro assay, specific for proteins, known in the art. The activity of the mammalian recombinant protein produced by Pseudomonas can be compared with the activity of the native mammalian protein, corresponding, to determine whether or not the mammalian recombinant protein shows activity substantially similar or equivalent to the activity generally observed in the native protein, under the same or similar physiological conditions. The activity of the recombinant protein can be compared with a standard activity of native protein, previously established. Alternatively, the activity of the recombinant protein can be determined in a simultaneous or substantially simultaneous comparative assay with the native protein. For example, in vitro assays can be used to determine any detectable interaction between a recombinant protein and an objective, for example, between an expressed enzyme and the substrate, between the expressed hormone and the hormone receptor, between the expressed antibody and the antigen, etc. Such detection may include measurement of colorimetric changes, proliferation changes, cell death, cell repulsion, changes in radioactivity, solubility, changes in molecular weight as measured in gel electrophoresis and / or exclusion methods in gel, phosphorylation abilities, antibody specificity assays such as ELISA assays, etc. In addition, in vivo assays include, but are not limited to, assays to detect the physiological effects of the protein produced by Pseudomonas in comparison to the physiological effects of the native protein, eg, weight gain, change in the balance of electrolytes, the change in the time of blood coagulation, changes in the dissolution of the clot, and the emulsion of the antigenic response. In general, any in vitro or in vivo assay can be used to determine the active nature of the mammalian recombinant protein produced by Pseudomonas that allows a comparative analysis of the native protein, as long as such an activity is assayable. Alternatively, the proteins produced in the present invention can be evaluated for the ability to stimulate or inhibit the interaction between the protein and a molecule that normally interacts with the protein, for example, a substrate or a component of the signal path with which Normally the native protein interacts. Such assays can typically include the steps of combining the protein with a substrate molecule, under conditions that allow the protein to interact with the target molecule, and to detect the biochemical consequence of the interaction with the protein and the target molecule. The assays that can be used to determine the activity of the protein are described, for example, in: Ralph, P.J., et al. (1884) J. Immunol. 1 32: 1 858; Saiki et al. (1981) J. Immunol. 127: 1 044; Steward, W. E. I I (1980) The Interferon Systems. Springer-Verlag, Vienna and New York; Broxmeyer, H. E., et al. (1 982) Blood 60: 595; Sambrook, J , E. F. Fritsch and T. Ma-niatis eds. (1 989) "Molecular Cloning: A Laboratory Manual", 2nd Ed. , Cold Spring Harbor Laboratory Press; Berger, S. L. and A. R. Kimmel eds. (1987) "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press; AK Patra et al. (2000) Protein Exp. Purif 1 8 (2): 1 82-92; Kodama et al. (1 986) J Biochem. 99: 1465-1472; Stewart et al. (1 993) Proc. Nat'l Acad. Sci. 90: 5209-521 3; Lombillo et al. (1,995) J. Cell Biol. 128: 1 07-1 15; Vale et al. (1 985) Cell 42: 39-50.

EXAMPLES Bacterial strains and growth conditions Unless otherwise specified, all strains used for all Pseudomonas expression tests were based on P. fluorescens strain MB 101. £. coli strains JM 1 09 (Promega), XL2 Blue (Stragene) or Tp 1 0 (Invitrogen) were used for cloning in general. For expression studies in £. coli, BL21 (DE3) Gold was used. Strains of P. fluorescens were developed either in the LB medium or in minimal salts supplemented with 1 5 μg / ml of tetracycline and 30 μg / ml of kanamycin as necessary at 30 ° C. The strains of £. coli were developed in LB supplemented with 30 μg / ml kanamycin and / or 1 5 μg / ml chloramphenicol, or 15 μg / ml tetracycline as necessary at 37 ° C. The cells were induced with 0.3 mM IPTG after the growth phase.

Detection of protein activity (ELISA assay) The plates were coated by the addition of 200 μl of β-galactosidase solution at 10 μg / ml in PBS (pH 7.6) to each well of the microtiter plate. The plates were incubated at room temperature for 16 hours, and then washed 3 times with 200 μl of PBS + 0.1% of Tween-20 (PBS-T). The primary antibody was diluted in PBS with 2% (w / v) skim milk powder. 200 μl of the diluted antibody was added to each well and incubated at room temperature for 1 hour. The plates were washed 4 times with 200 μL of PBS-T. The secondary antibody was also diluted in PBS with 2% skimmed milk powder (w / v) and 200 μL was added to each well, and incubated at room temperature for 1.5 to 2 hours. The plates were then washed 4 times with PBS-T. A tertiary antibody is used to detect scFv antibodies: sheep anti-mouse antibody, conjugated alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA cat # A5324). To each desired well was added 200 μL of the diluted antibody solution (or PBS-T) and incubated at room temperature for 1.5 ho'Vas. The plates were then washed 4 times with PBS-T. 200 μl of the freshly prepared Sigma Fast pNPP substrate (Sigma catalog # R-2770) was added to each well. After 30 minutes, the reaction was stopped by the addition of 50 μL of 2M sodium hydroxide to each well, and the absorbance was read at 405 nm.

Fermentation. The inoculum for the culture of the fermenter for P. fluorescens is generated by the inoculation of a shake flask containing 600 ml of chemically defined medium supplemented with yeast extract and dextrose. Tetracycline is typically added to accelerate the maintenance of the recombinant plasmid in the starter culture during its overnight incubation, as well as in the fermenter. The flask culture is then aseptically transferred to a 20 liter fermenter containing a chemically defined medium designed to withstand a high biomass, without supplementation with yeast extract. Oxygen is maintained at a positive level in the liquid culture by regulating air flow in the fermenter and mixing speed of the agitator; the pH is maintained at plus 6.0 through the addition of aqueous ammonia. The fermentation process at high feed batch density is divided into an initial growth phase of approximately 24 hours and the gene expression phase (induction) in which an inducer is added to initiate the expression of the recombinant gene. Glucose, in the form of corn syrup, is fed throughout the fermentation process at limiting concentrations. The density of the target cells to initiate the induction phase is typically 1 50 OD units at 575 nm. The induction date of the fermentation is typically allowed for approximately 45 to 55 hours. During this phase, samples are taken from the fermenter for various analyzes, to determine the level of expression of the target gene, cell density, etc. For each fermentation experiment for £. coli, a frozen storage glycerol stock at .80 ° C, thawed diluted before inoculation of a shake flask containing 600 ml of LB broth supplemented with kanamycin, is removed. The shake flask culture is incubated at 37 ° C with shaking at 300 rpm overnight and then aseptically transferred to a 20 liter fermentor containing the complete medium. The temperature of the fermenter is maintained at 37 ° C, the pH 7 through the addition of aqueous ammonia and phosphoric acid and dissolved oxygen to more than 20%. After a brief initial phase of the batch, the glycerol is fed at increasing speeds to keep excess carbon. When the cell density reaches 24-28 OD units at 600 nm, recombinant expression is effected by the addition of an inducer, such as isopropyl thiogalactoside (IPTG). The induction phase of the fermentation typically continues for approximately 3 to 5 hours as the fermenter reaches the volumetric capacity or as the growth rate begins to decrease significantly. During this phase, the samples are removed from the fermenter for various analyzes, to determine the level of expression of the target gene, cell density, etc.

Cell fractionation and analysis of SDS-PA GE The samples are normalized to A575-30, and 1 ml of the normalized culture is concentrated by centrifugation. The cells are resuspended in 1 ml of lysis buffer (50 mM Tris base, 200 mM sodium chloride, 5% v / v glycerol, 20 mM EDTA disodium salt, 0.5% Triton X-1 00 v / v; DTT 1 mM). A specific protease inhibitor cocktail is added to the bacterial lysates (Sigma # P8465) at a concentration of IX. The resuspended cells are added to a microcentrifuge tube with a screw cap of 2 ml, approximately at%. filled with 0. 1 mm glass spheres and the cells are mechanically lysed using 4 1 minute incubations in a BioSpec sphere mill at the highest setting. The cells are kept on ice between incubations. Approximately 1000 μL of the lysed cell solution are removed from the spheres, transferred to a new tube and concentrated by centrifugation. The supernatant (soluble fraction) is withdrawn into a new tube. The button (insoluble fraction) is resuspended in an equal volume of 1 00 μl of the lysis buffer plus the protease inhibitor. Five μL of each sample were added to 5 μL of the 2X LDS charge buffer (Invitrogen) and loaded onto a Bis-Tris NuPAGE gel of 4-12% or 10% (Invitrogen) and run either in IX MES or IX buffer MOPS as indicated.

Example 1. Expression of scFV in the cytoplasm Single-chain antibody fragments (scFV) are found to have increased use as diagnostic and therapeutic agents. These relatively small proteins are made by fusion of genes encoding the heavy and light chains of an immunoglobulin.

Cloning of Gall3 scFV The Gall3 scFv gene (Genbank accession number AF238290), cloned into phage display vector pCANTABd, (see P Martineau et al. (1998) J. Mol. Biol. 280: 1 1 7- 27) was used as a template to amplify a product of 774 base pairs, which was subsequently cloned into the pCR2.1 TOPO vector (Invitrogen, Carisbad, CA, USA). The scFv gene was excised from the TOPO vector with the restriction enzymes Spel and Salí (New England Biolabs, Beverly, MA, USA) and cloned into the Xpel and Xhol sites of the pMYC 1 803 vector of P. fluorescens, downstream ( 3 ') of the Ptac promoter, to produce pDOW 1 1 1 7. The resulting plasmids were sensed in electroporesis in P. fluorescens. The Gall3 gene was cloned into the expression vector pET24d + (Novagen, Madison, Wl, USA), after amplification, such that the Sali and Ncol sites flanked the coding sequence. The PCR products were digested with Sali and Ncol, and cloned into the same sites of the pET24d + vector downstream (3 ') of the T7 promoter. The newly formed construction was then used to transform the XL2 blue component cells. Once the sequence was confirmed, the DNA construct was used to transform BL21 (DE3) Gold (Stratagene, San Diego, CA, USA) for expression.

Expression of single chain antibody fragments (scFv) in E. coli and P. fluorescens The scFv molecules were expressed in £. coli and P. fluorescens, including a scFv with binding activity to the single-chain antibody gal 13 of the β-galactosidase protein of £. coli (P. Martineau et al., "Expression of an antibody fragment at high levéis in the bacterial cytoplasm," J. Mol. Biol. 280 (1): 1 1 7-27 (1,998)). P. fluorescens expressed approximately six times more protein than £. coli than the fermentation of 20 liters, with a yield of 3.1 g / liter in P. fluorescens and 0.5 g / liter yield in £. coli, as determined by SDS-PAGE and densitometry (see Table 8). P. fluorescens was expressed approximately 96% soluble protein, while £. coli expresses only 48% soluble protein.

TABLE 8: Summary of Gal13 fermentation (* compared to BSA standards) The material purified from the two expression systems was found to be active in an immunosorbent assay enzyme-linked (ELISA) as shown in Figure 2. The material was also purified from the soluble fraction only of volumes used equal to from solubilized from both strains, using as affinity chromatography. Finally, the complete volumetric recovery for the P. fluorescens process is approximately 200 times more efficient than for £. coli, 1.34 g / liter against 0.07 g / liter.

Example 2: Expression of human? -IFN in the cytoplasm Cloning of human gamma-interferon The human interferon gamma (hu-? LFN, Genbank access X13274) was amplified from a human spleen cDNA library (Invitrogen, Carisbad, CA, USA; catalog # 1 0425-01 5) such that it lacked the native secretion signal at the N-terminus of the recombinant? -I FN that it begins as Met-Cys-Tyr-Cys-Gln-Asp-Pro as described in PW Gray et al. (1 982) Nature 298: 859-63. The resulting product was cloned into the pCR2.1 TOPO vector and the sequence was confirmed. The hu-? Fn gene was excised from the TOPO vector with the restriction enzymes Spel and Xhol and cloned into the same sites of pMYC1 803. In a separate reaction, hu-? FN was amplified such that the Afl lly and Xhol sites they flanked that coding sequence. The resulting fragment was cloned into the TOPO-TA vector (Invitrogen) and transformed into £ cells. coli JM1 09 which are technically competent (Promega, Madison, Wl, USA). The gene was isolated by digestion with Afll lly Xhol (New England Biolabs), cloned into the Ncol and Xhol sites of pET24d + (Novagen, Madison, Wl, USA) downstream (3 ') of the T7 promoter, and transformed into J M1 09. A positive clone was transformed inside the £ cells. coli BL21 (DE3) (Novagen) to test the expression.

Purification of human gamma-interferon The frozen cell paste from P. fluorescens cultures was thawed and re-suspended in lysis buffer (50 mM potassium phosphate, pH 7.2 containing 50 mM sodium chloride, 10 mM EDTA (ethylenediaminetetraacetic acid) , catalog number BPII8-500, Fisher Scientific, Springfield, NJ, USA), 1 mM PMSF (phenylmethylsulfonyl fluoride, catalog number P-7626, Sigma, St. Louis, MO), (catalog number D-0632, Sigma), and 1 mM benzamide (catalog number B-6506, Sigma)) at a ratio of about 1 g of cell paste with 2 ml of lysis buffer. The cells were disintegrated by three steps through a microfluidizer (model 1 1 0Y, Microfluidics Corporation, Newton, MA, USA). The cell roof and the non-disintegrated cells were removed by centrifugation (for 60 minutes at 23.708x g and 4 ° C using a Beckman Coulter centrifuge, model JA 25.50, Coulter, Inc., Fullerton, CA, USA). The resulting supernatant (cell-free extracts) was clarified by addition of diatomaceous earth to 1 0% w / v (Celite product, World Minerals, Inc., Goleta, CA, USA) and passing the result through a paper filter (Whatman 1, catalog number 1 001 -150, Whatman Paper Ltd., Maidstone, Kent, United Kingdom)) with vacuum filtration.

Clarified cell extracts were applied to a 3.2 cm x 1 3.5 cm chromatography column of SP-Sepharose FAST FLOW (6% cross-linked agarose gel material); catalog number 17-0709-1 0, Amersham Biosciences, Piscataway, NJ, USA) equilibrated in buffer A, at a flow rate of 0.5 ml / minute. The buffer composition was: 50 mM HEPES, pH 7.8 (for example, N- (2-Hydroxyethyl) piperazine) -N '- (2-ethanesulfonic acid), Fisher Scientific, catalog number BP-310-100) chloride 50 mM sodium, 1 mM EDTA, and 0.02% sodium azide (catalog number 71 289, Sigma Chemical Co.). After loading, the column was washed with 3 column volumes (column volume = January 08 ml) buffer to and 5 column volumes of buffer A containing cioruro sodium 0.4 M. The column was subsequently revealed by applying a gradient of 0.4 M to 1 M sodium chloride in the same buffer, at a flow rate of 2 ml / minute for a total of 7 column volumes. Fractions that contained I FN-? were then combined and dialysed against 1 X PBS (buffered saline, pH 7.2) at 4 ° C. The protein was concentrated by ultrafiltration (using a YM30 ultrafiltration membrane; catalog no. 13722, Millipore, Bedford, MA USA), then frozen in liquid nitrogen and stored at 80 ° C.

Expression of human β-interferon in E. coli and P. fluorescens The human α-interferon is commercially produced by fermentation of £. coli expressing the gene? -I FN. The protein is expressed cytoplasmically in a soluble and inactive form. In order to produce the recombinant polypeptide as an active pharmaceutical ingredient, the interferon must be recovered as solubilized, refolded, and then purified. All of these unit operations are largely added to the cost of goods (COGs) for this protein. A human spleen cDNA library was used as a template to amplify the? FN cDNA without the native signal sequence and the clone within the £ expression vectors. coli and P. fluorescens. The construction of P. fluorescens produced - 4 g / liter of the protein? LFN during a typical fermentation reaction of 20 liters. The SDS-PAGE and Western analyzes of the insoluble and soluble fractions show that most of the protein (95%) is present in the soluble fraction. Figure 1 shows that hu -? - I FN purified from the soluble fraction of P. fluorescens samples shows activity comparable to a commercially available standard. Figure 5 and Table 9 show a comparison of the expression of? -I FN between the expression systems of £. coli and P. fluorescens.

TABLE 9: Fermentation Summary of? -IFN (* compared to BSA standards) Human gamma-interferon activity assay Cell lines and media: Hela cells (catalog No. CCL-2) and encephalomyocarditis virus (ECMV, catalog No. VR-129B) were obtained from the American Type Culture Collection (Manassas, GOES). HeLa cells were maintained in Eagle's modified essential medium (Cellgro EMEM, VA, USA) with 10% bovine fetal serum (Gibco, Invitrogen, Carisbad, CA, USA) at 37 ° C / 5% C02. The activity of hu-? L purified FN was evaluated using a viral inhibition assay as previously described (JA Lewis (1987) in Lymphokines and Interferons: A Practical Approach MJ Clemens et al. (Eds.) (I RL Press Ltd , Oxford, England.) In summary, the HeLa cells were seeded in a 96-well microtiter plate at 3 x 1 04 per well.After 24 hours, the purified hu-? FN isolated from P. fluorescens, or the human The recombinant E. coli FN (from R &D Systems, Minneapolis, USA), were added to wells in triplicate at 0, 0.001 or 0.05 ng per well, after preincubation of the cells with hu-? l FN for 24 hours, ECMV was added to variant dilutions to the groups of wells in triplicate.The cells were incubated for 5 days, after which the cell viability was measured using an ELI SA of cell proliferation that monitored the incorporation of Bromo-2'-deoxyuridine (catalog No. 1647229, Roche Molecular Biochemic als, Ind ianapolis, I N, USA). The results are expressed as absorbance units, with higher absorbance resulting from the presence of a larger number of cells that are actively dividing (life).

Example 3: Expression of hGH in the cytoplasm Primers were designed to amplify human growth hormone (hGH) from human cDNA libraries. For this study, hGH was amplified using the AmpliTaq polymerase (Perkin Elmer) according to the manufacturer's protocol, using the above plasmid as a template and the ELVIrev and hgh-sig primers, with a PCR cycle profile of 95 ° C per 2 minutes (95 ° C, 60 seconds, 42 ° C, 120 seconds, 72 ° C, 3 minutes) 25X. The resulting product was purified using the Wizard PCR DNA purification kit (Promega), digested with the Sepl and Xhol restriction enzymes (New England Biolabs) and cloned into the same site of pMYC1 803 (see Figure 3). A mutation found in amplified hGH was corrected by using primer hgh-sigcorr with ELVirev and repeating the PCR and cloning procedures. Primers used to clone hGH. hGH- AGAGAACTAGTAAAAAGGAGAAATCCATGTTCCCAACCATT sig CCCTT ATC HGH- AGAGAACTAGTAAAAAGGAGAAATCCATGTTCCCAACCATT sigcorr CCCTT ATCCAGGCCTTTTGAC ELVIfor AGAGAACTAGTAAAAAGGAGAAATCCATGGCTACAGGCTCC CGGA CGTCC ELVIrev AGAGACTCGAGTCATTAGAAGCCACAGCTGCCCTCCAC Purification of hGH After fermentation in 20 liters, hGH was purified from the insoluble fraction of cells of £. coli and P. fluorescens, with the exception that during the elution of DEAE FF, a gradient of 0 to 0.5 M sodium chloride was used instead of the sodium chloride step 0. 25 M.

Expression of human growth hormone in E. coli versus P. fluorescens The cDNA encoding human growth hormone was purified from a human pituitary cDNA library. The secretion signal sequence was removed, and an N-terminal methionine was engineered into constructs for microbial expression. - For the expression in £. coli, the pET25 vector containing the hGH gene was transformed into BL21 (DE3), which contains an integrated T7 polymerase gene, necessary for the transcription of hGH. Expression studies in P. fluorescens were carried out in strain MB214, which contains an integrated lacl gene, to control expression from the Ptac promoter. Both expression systems were evaluated at the fermentation scale of 20 liters. As shown in Table 10, P. fluorescens (Pf) worked better than £. coli (EC) in the amount of protein produced per gram of dry biomass (1 .6X more).

TABLE 1 0: Summary of hGH fermentation (^ compared to BSA standards) The SDS-PAG E cell fractionation analyzes show that hGH is found in the insoluble fraction in both expression systems (figure 4). Surprisingly, approximately 7X more monomer hGH was purified from P. fluorescens, in comparison to £. coli, despite a difference of only 1.6X in the production of protein per gram of dry biomass.

TABLE 1 1: Comparison of the purification of hGH from E. coli and P. fluorescens Example 4: Expression of proteins in the periplasm Characterization of secretion signal peptides Secretion signal objects of Pseudomonas fluorescens were studied by the formation and expression of fusions of genomic DNA - sequence encoding alkaline phosphatase (phoA) and are described in more detail in U.S. Application No. 1 0 / 996,007, filed on November 22, 2004. Six of the mergers expressed were further characterized as follows. The cleavage site for the signal sequences for the secreted genes identified with phoA fusions were deduced by comparison to the homologous proteins from another Pseudomonas, by the SPScan program (Menne et al, 2000). The cleavage site of the putative lipoprotein was deduced by comparison to the peptidase portions of I I signal; signal peptidase I I specifically breaks down the signal sequences of lipoproteins. The six signal peptides were analyzed using SignalP (a software program for the analysis of putative signal peptides available from the Center for the Analysis of Biological Sequences of the Technical University of Denmark, at http://www.cbs.dtu .dklservices / SignalP /.) See also, Nielson et al. (1 997) Protein Engineering 1 0: 1-6. In some cases, a supplemental source was used to further characterize the identity of the signal peptide. This information is present in table 1 2.

TABLE 1 2: Identity of secretion signal peptides Western analysis of the phoA fusion proteins to detect the fusion proteins To analyze the fusion proteins were produced, Western analysis with alkaline phosphatase antibody was carried out on separate cultures by centrifugation in a whole cell fraction ( cytoplasm and periplasm) and a fraction of cell-free broth. Of five strains for which the insertion site was determined, four (putative azurine, putative phosphate binding protein, putative periplasmic lipoprotein B, putative protein binding to Fe (III)) produced a fusion protein of the expected size, and one (putative oprE protein) produced a protein approximately 40 kD smaller than predicted, and one (putative Lys-Arg-Orn binding protein) produced a protein approximately 20 kD smaller than predicted. The proteins were separated by SDS-PAGE and transferred to the microcellulose membranes at 40 V for one hour using the Xcell SureLockTM Mini-Cell and XCell 11 TM Blot (Invitrogen). The Western experiments were performed using the instruction provided by the SuperSignal West HisProbeTM (Pierce) team.Construction, expression and characterization of a pbp-hGH fusion The phosphate-binding protein secretion guide of P. fluorescens was fused to the N-terminus of the mature domain of the human growth hormone (hGH) gene and tested for expression and the secretion The coding region of the pbp signal sequence was amplified by PCR from a clone of the pbp signal sequence of P. fluorescens as a template using sig_pbp for (gctctagagggaggtaacttatgaaactgaaacg) and pbp_hgh (gggaatggttgggaaggcccaccgcgttggc), as primers, and then purified in gel. This resulted in the production of an oligonucleotide fragment containing the pbp CDS signal peptide and the coding sequence for the 5 'end of the mature hGH domain. A cDNA encoding human growth hormone was amplified by PCR from the human pituitary cDNA library (Clontech, Palo Alto CA) using primers ELVIfor (agagaactagtaaaaaggagaaatccatggctacaggctcccggacgtcc) and ELVIrev (agagactcgagtcattagaagccacagctgccctccac), which were designed to amplify only the mature domain of hGH, and cloned into pMYC1 803 / Spel Xhol, forming pDOW2400. The mature hGH gene was amplified from pDOW2400, using primers pbp_hgh_revcomp (gccaacgcggtggccttccaaccattccc) and hgh_rev (agagactcgagtcattagaagccacagctgcctccacagagcggcac), and then purified with columns (Stratagene) to remove the primers and other reaction components. To make the polynucleotide that codes for the pbp-hGH fusion, the two PCR reactions were combined and amplified again with sig_pbp for and hgh_rev in order to ligate the two pieces. The expected fragment of 681 base pairs was purified with Strataprep as mentioned above, restriction digested with Xbal and Xhol and ligated to pDOW 1 269 / dephosphorylated XholSpel, to form pDOW 1 323-1 0, placing pbp-hGH under control of the tac promoter in an analogous vector pMYC-1 1 803, but with a selectable marker by pyrF in place of a tetR tetracline resistance marker gene. The ligature mixture was transformed into MB1 01 pyrF proC laclQ1. The inserts were sequenced by the Dow Chemical Company using the method described above. The DNA and amino acid sequence of this fusion is presented in Figure 10 and Figure 11, respectively. The resulting strains were first tested on the scale of the shake flask. Induced bands of the expected size for processing and unprocessed (22.2 kDa and 24.5 kDa, respectively) were detected by SDS-PAGE.

Approximately half of the proteins were processed (indicating the location towards the periplasm), and of the processed one, approximately half was in the soluble fraction and half in the insoluble fraction. Expression studies were elevated in scale to 20-liter bioreactors. Densitometry of Coomassie-stained SDS-PAGE genes showed that 1 8% of the total hGH produced was processed and soluble. The productive strain 3.2 g / liter of all forms of hGH; processed and soluble was 0.6 g / liter.

Construction, expression and characterization of pbp-scFv fusion The putative 24-amino acid signal sequence of the phosphate-binding protein (eg, including Meti) was fused to the open reading structure of the scFv gene of gal2 (gal2) ) at the position of the amino acid +2 (Ala). See figure 8 and figure 9. The signal sequence seems to be processed, indicating secretion towards the periplasm. In addition, there is secretion towards the broth, since the protein was detected in the supernatant of cell-free culture. Surprisingly, fusion to the signal sequence of the phosphate binding protein appears to improve scFv expression of gal2 in P. fluorescens. Without the secretion signal fused at the amino terminus, scFv expression of gal2 was not detectable.

Cloning of gal2 PCR was performed using the primers sig_pbp for (previously mentioned) and pbp_gal2SOE rev (ctgcacctgggcggccaccgcgtt) containing a reverse complement of pbp__tgaI2SSOE for (aaccgcggtggccgcccaggtgcag) and using a plasmid encoding the pbp secretion signal peptide of P. fluorescens as a template. This resulted in the production of an oligonucleotide fragment containing the coding sequence of the pbp signal peptide (CDS) and a CDS for the 5 'end of the gal2 single chain antibody (scAb or scFV). The PCR is related using primers pbp_gal2SOE for and scFv2rev (acgcgtcgacttattaatggtg atgatggtgatgtgcggccgcagttgatc), and a polynucleotide coding for gal2 was used as a template. This resulted in the production of a polynucleotide fragment containing a CDS encoding the 3 'end of the pbp signal peptide and the open reading frame (ORE) encoding gal2. The reaction products were purified. Approximately 15 ng of each were used as an AD N "template" in an additional PCR reaction using the primers sig_pbp_f r and scFV2rev. This resulted in the production of a nucleic acid fragment with the pbp signal sequence, CDS fused to the gal2 coding sequence. The predicted amino acid -1 of the signal sequence (ie, the last amino acid before the proposed cleavage site) was fused to the +2 amino acid of scFv of gal2 (Ala). The resulting fusion was cloned into the vector pMYC1 803 of P. fluorescens under the control of the Ptac promoter to produce the plasmid and pDOW1 123 (pbp: gal2). The plasmid was transformed into strain P.sub.1 01 of P. fluorescens carrying the plasmid pCN51-lacl (described in United States Application No. 10/994, 138, filed November 1, 2004.

Fusion of the signal sequence of the putative phosphate binding protein for scFv of gal2 The signal sequence of the phosphate binding protein was fused to a single chain antibody gene and tested for secretion into the periplasm and / or the culture supernatant.

TABLE 1 3: Summary of fermentation of secreted Gal2 (* compared to BSA standards) The resulting strains were first tested on the shake flask scale. Induced bands of the expected size for unprocessed and processed gal2 (29 kDa and 28 kDa) were detected via the SDS-PAGE in the insoluble protein fraction (data not shown). The expression studies were elevated from scale to fermentation of 20 liters. Again, SDS-PAGE analysis showed that most of the induced proteins were found in the insoluble protein fraction. Western analysis also indicated that some processed gal2 is present in the soluble protein fraction for pbp: gal2 (pDOW 1 123). Western analysis of the periplasmic fractions prepared from the strains possessing pDOW 123 (using the epicenter periplasmic team) showed the presence of the soluble gal2 protein. Recombinant gal2 scFv was isolated from the cell extract of a shake flask experiment using a Quiagen Ni-NTA protocol, then refolded as described in Martineau et al. , J Mol. Biol. 280: 1 17-127 (1,998). This antibody was found to be active against β-galactosidase in an ELISA assay.

Claims (30)

1. RE IVI NDICATIONS 1 . A process for increasing the expression of a mammalian recombinant protein, comprising: a. Transformation of a host cell of Pseudomonas fluorescens with a nucleic acid encoding some mammalian recombinant protein; and b. developing the cell under conditions that allow the expression of the mammalian recombinant protein; wherein the protein is expressed at an increased level as compared to a level of expression of the protein under substantially comparable conditions in a £ expression system. coli
2. 2. The process according to claim 1, which further comprises the isolation of the mammalian recombinant protein.
3. 3. The process according to claim 2, further comprising substantially purifying the mammalian recombinant protein.
4. 4. The process according to claim 1, wherein the mammalian recombinant protein is present in the host cell in a soluble form.
5. 5. The process according to claim 1, wherein the mammalian recombinant protein is present in the host cell in an insoluble form.
6. 6. The process according to claim 1, wherein the mammalian recombinant protein is present in the host cell in an active form.
7. 7. The process according to claim 1, wherein the mammalian recombinant protein is a human peptide.
8. 8. The process according to claim 1, wherein the recombinant protein is produced as more than about 5% of the total cellular protein.
9. 9. The process according to claim 1, wherein the recombinant protein is produced as more than about 10% of the total cellular protein.
10. 10. The process according to claim 1, wherein the recombinant protein is produced at a concentration of at least 10 g / liter. eleven .
11. The process according to claim 1, wherein the recombinant protein is produced at a concentration of at least 20 g / liter.
12. 12. The process according to claim 1, wherein the recombinant protein is produced at a concentration of at least 40 g / liter.
13. 13. A process for producing a mammalian recombinant protein in a host cell of Pseudomonas fluorescens, which comprises: a. transforming a host cell with a nucleic acid coding for a mammalian recombinant protein; b. develop the cell under conditions that allow the expression of the mammalian recombinant protein, and c. isolate the mammalian recombinant protein.
14. 14. The process according to claim 13, further comprising substantially purifying the mammalian recombinant protein.
15. 5. The process according to claim 13, wherein the mammalian recombinant protein is present in the host cell in a soluble form.
16. The process according to claim 13, wherein the mammalian recombinant protein is present in the host cell in an insoluble form.
17. 17. The process according to claim 13, wherein the mammalian recombinant protein is present in the host cell in an active form.
18. 18. The process according to claim 13, wherein the mammalian recombinant protein is a human peptide.
19. 9. The process according to claim 13, wherein the mammalian recombinant protein has a mass of at least about 1 kD and about 500 kD.
20. 20. The process according to claim 19, wherein the mammalian recombinant protein has a mass greater than about 30 kD. twenty-one .
21. The process according to claim 1, wherein the recombinant protein is produced as more than about 5% of the total cellular protein.
22. 22. The process according to claim 1, wherein the recombinant protein is produced as more than about 10% of the total cellular protein.
23. 23. The process according to claim 13, wherein the recombinant protein is produced at a concentration of at least 10 g / liter.
24. 24. The process according to claim 1 3, wherein the recombinant protein is produced at a concentration of at least 20 g / liter.
25. 25. The process according to claim 13, wherein the recombinant protein is produced at a concentration of at least 40 g / liter.
26. 26. A process for producing a recombinant human protein in a host cell comprising: a. transforming the host cell with a nucleic acid encoding a recombinant human peptide; and b. develop the cell under conditions that allow the expression of the recombinant human peptide; wherein the host cell is Pseudomonas fluorescens.
27. 27. The process according to claim 26, wherein the recombinant human protein is present in soluble form in the host cell.
28. 28. The process according to claim 26, wherein the recombinant human protein is present in active form in the host cell.
29. 29. A Pseudomonas fluorescens cell, comprising a nucleic acid encoding a recombinant human peptide.
30. 30. The cell according to claim 29, wherein the recombinant human peptide is expressed.