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Patent Application Titled "Constructs and Methods for the Production and Secretion of Polypeptides" Published Online

August 28, 2014



By a News Reporter-Staff News Editor at Politics & Government Week -- According to news reporting originating from Washington, D.C., by VerticalNews journalists, a patent application by the inventors FIEROBE, Henri-Pierre (Marseille, FR); Mingardon, Florence (Pasadena, CA); Chanal-Vial, Angelique (Marseille, FR), filed on April 9, 2014, was made available online on August 14, 2014.

No assignee for this patent application has been made.

Reporters obtained the following quote from the background information supplied by the inventors: "Secretion of heterologous proteins is a widely used technique in industry. A cell can be transformed with a nucleic acid encoding a heterologous protein of interest to be secreted and thereby produce large quantities of desired proteins. This technique can be used to produce a vast amount of protein over what would be produced naturally. Proteins of interest are proteins with a wide variety of industrial applications, including therapeutic and agricultural uses, as well as use in foods, cosmetics, cleaning compositions, animal feed, etc. Thus, increasing secretion of proteins produced by micro-organism is of general interest.

"Advances in cellular and molecular biology have made it possible, in certain cases, to identify a gene encoding a desired protein, to isolate the gene, to insert the gene into a host cell and to express the inserted gene in the host cell to produce the desired protein. Bacteria have been intensively studied as host cells. When bacteria are used as host cells for this heterologous gene expression, a frequently encountered problem is however that most bacterial expression systems produce proteins intracellularly, and it is usually necessary to disrupt the cells to ensure recovery of the products.

"The problem may be overcome by having the bacteria secrete the desired protein into the growth medium. One particularly well documented method of directing the secretion of proteins is the use of a secretory signal sequence. When a signal peptide is fused to the amino-terminal end of a heterologous protein, it directs the heterologous protein to the secretory machinery at the cell membrane. The heterologous protein is then translocated across the membrane. Optionally a specific protease, sometimes referred to as 'signal peptidase' or 'leader peptidase', removes the signal peptide and releases the heterologous protein.

"Translocation of proteins into periplasmic space or secretion into their culture media is subject to a variety of parameters. Typically, vectors for secretion of a protein of interest are engineered to position DNA encoding a secretory signal sequence 5' to the DNA encoding the protein of interest. To increase secretion several approaches can be followed: trying several different signal sequences, mutating the signal sequence, or altering the secretory pathway within the host. However, in many cases the amount of heterologous protein secreted when making use of only a signal peptide to ensure secretion is usually very small, and a significant amount of the heterologous protein is often degraded after it is secreted.

"Clostridium is a genus of Gram-positive bacteria, which is represented by a wide variety of strains. Clostridium bacteria are spore-forming anaerobic bacteria. This genus comprises solventogenic Clostridia such as C. acetobutylicum that are able to convert various sugars and polysaccharides into acids and solvents, and cellulolytic Clostridia, such as Clostridium cellulolyticum, that are able to efficiently degrade cellulose and related plant cell wall polysaccharides. More in particular, Clostridium cellulolyticum produces and secretes large cellulolytic complexes called cellulosomes that efficiently degrade cellulose and related plant cell wall polysaccharides. These complexes contain various enzymes which are tightly bound to a large protein devoid of enzymatic activity called 'scaffoldin'. The binding of the enzymes on the scaffoldin occurs through interaction between cohesion modules on the scaffoldin and complementary dockerin domains on the enzymes. This high affinity interaction between dockerins and scaffoldins has been suggested for biotechnology applications e.g. recombinant protein purification (Craig et al. 2005, J. Biotechnol. 121:165-173).

"On the contrary, C. acetobutylicum although it contains in its genome contains a large cluster of genes encoding cellulolytic enzymes and a scaffoldin, is not able to grow on crystalline cellulose.

"One of the strategies to combine cellulose-degrading activity with solvent production in one organism has been to introduce the genes encoding the cellulosome of C. cellulolyticum into C. acetobutylicum. Mingardon et al. have demonstrated the production, assembly and secretion of a minicellulosome by Clostridium acetobutylicum by co-expressing the Mannanase gene Man5K from Clostridium cellulolyticum with the gene cipC1 encoding a truncated scaffoldin also from C. cellulolyticum therein (Mingardon et al. Applied Environm. Microbiol. 2005, vol 71(3): 1215-1222).

"Several groups have investigated the possibility of increasing or improving the cellulolytic activity of cellulosome complexes by playing with the different modules present therein and combining different types of cellulases in what is referred to as 'designer cellulosomes'. It was demonstrated that bifunctional and trifunctional designer cellulosomes which include a chimeric scaffoldin with two or three cohesins of divergent specificity and two or three cellulases each bearing a dockerin complementary to one of the cohesins yielded a multiprotein complex with enhanced synergistic activity on recalcitrant substrates such as straw (Fierobe et al. 2002, J. Biol. Chem. 277, 49621-19630; Fierobe et al. 2005, J. Biol. Chem. 280(16):16325-16334). In addition it was found that such cellulosomes could include combinations of bacterial and fungal enzymes (Mingardon et al. 2007, Appl. Environm. Microbiol. 73(12):3822-3832). In these experiments the cellulosomes were either produced by co-expression of the vectors encoding the different parts of the cellulosome in Clostridium cellulolyticum which naturally secretes these proteins or by mixing the recombinantly produced and purified scaffoldins and enzymes in vitro.

"Mingardon et al. describes the production of a 'covalent cellulosome', which comprises, in a single polypeptide chain, a CBM together with a family 48 and a family 9 catalytic module. This protein was recovered from E. coli in which it was overexpressed by breaking the cells in a French press and purifying the recombinant protein using the c-terminal His tag. The covalent cellulosome was found to be significantly less active on Avicel substrate than the corresponding hybrid cellulosomes (Mingardon et al. 2007, Appl. Environm. Microbiol. 73(22):7138-7149).

"Cloning of heterologous or homologous genes encoding secreted proteins, and (over)production and secretion of such heterologous or homologous proteins by bacterial cells such as Clostridium species other than C. cellulolyticum has not been very widely reported up until now, probably as a result of problems encountered with ensuring secretion of recombinant proteins by these hosts.

"In view of the above, it is clear that there is a need in the art to improve secretion of proteins by bacterial cells."

In addition to obtaining background information on this patent application, VerticalNews editors also obtained the inventors' summary information for this patent application: "The instant invention aims to provide an approach to produce and secrete heterologous polypeptides of interest by a bacterial cell, more particularly a gram-positive bacterial cell and/or to improve the production and secretion of homologous polypeptides of interest by a gram positive bacterial cell, and in particular in a Clostridium bacterium. Also provided herein are novel molecules and constructs useful in the methods of protein secretion provided herein, and methods of making such molecules and constructs.

"The present application is at least partly based on the finding of a new method for microbial production and export of a polypeptide of interest which avoids at least some of the problems associated with secretion as enumerated above. The molecules, constructs and methods according to this invention make it possible to (over)produce and secrete polypeptides of interest, by a bacterial cell. In particular, the present invention provides a polynucleic acid encoding a fusion protein, wherein said fusion protein has a carrier domain which has a functional effect on the secretion of the fused polypeptide of interest. More in particular, the inventors have shown a functional effect of a carrier domain of a fusion protein, i.e. the capability of controlling (inducing and/or improving) (extracellular) secretion of a homologous or a heterologous polypeptide of interest by a recombinant host cell, producing said fusion protein. Said carrier domain comprises a carbohydrate binding module (CBM) and a hydrophilic module (X module), typically of a scaffolding protein and, more particularly in combination with a secretion signal peptide ensures (improved) secretion of a polypeptide of interest. As such the present invention thus advantageously also provides for the use of at least a part of a scaffolding protein, and in particular at least the modules including a CBM, a hydrophilic module thereof, in particular in combination with a signal peptide, for controlling secretion in a host cell of a homologous or a heterologous polypeptide of interest fused to said part of the scaffolding protein.

"In a first aspect, the invention therefore provides a polynucleic acid encoding a fusion protein consisting of a polypeptide sequence which comprises in this particular order: a carrier domain comprising at least one carbohydrate binding module (CBM) of a cellulosomal scaffolding protein fused to at least one hydrophilic domain of a cellulosomal scaffolding protein; at least one peptide linker for linking the carrier domain to the polypeptide of interest, and at least one polypeptide of interest.

"In a particular embodiment of the invention said polynucleic acid further comprises an in frame nucleic acid sequence for the secretion of the encoded fusion protein, and preferably said nucleic acid sequence encodes a signal peptide of a cellulosomal scaffolding protein.

"Accordingly, the invention provides polynucleic acids encoding a fusion protein consisting of a polypeptide sequence which comprises, and more particularly, in this order: at least one suitable signal peptide a carrier domain comprising at least one carbohydrate binding module (CBM) of a cellulosomal scaffolding protein fused to at least one X module of a cellulosomal scaffolding protein; at least one polypeptide of interest, and at least one peptide linker for linking the carrier domain to the polypeptide of interest.

"In particular embodiments of the invention the peptide linker comprises a protease cleavage site for the cleavage of said polypeptide of interest from the remaining fusion protein.

"In further particular embodiments, the polypeptide sequence comprises two or more X modules, more particularly two X modules.

"In another aspect, the invention is directed to the use of a carrier domain as defined herein, more particularly in combination with a signal peptide, for controlling the secretion of a polypeptide of interest, preferably a polypeptide as defined herein, by a host cell.

"In another aspect, the present invention relates to a vector comprising a polynucleic acid according to the invention. Preferably a vector is provided wherein the polynucleic acid is under the control of regulatory sequences for expression of the nucleic acid in a bacterial cell.

"In yet another aspect, the invention provides a host cell comprising a polynucleic acid or a vector according to the invention.

"Accordingly, particular embodiments of the invention relate to recombinant micro-organisms comprising a polynucleic acid encoding a fusion protein consisting of a polypeptide sequence which comprises, more particularly in this order: (1) at least one signal peptide; (2) a carrier domain comprising at least one carbohydrate binding module (CBM), of the type of CBM of a cellulosomal scaffolding protein, fused to at least one X module of a cellulosomal scaffolding protein; (3) at least one polypeptide of interest; and (4) at least one peptide linker for linking the carrier domain to the polypeptide of interest. The micro-organisms of the invention are characterized in that they secrete the polypeptide of interest.

"In further particular embodiments, micro-organisms are provided wherein the polynucleic acid encodes a polypeptide sequence which comprises two or more X modules.

"In particular embodiments, micro-organisms are provided wherein the polypeptide sequence comprises a signal peptide, which is a signal peptide of a cellulosomal scaffolding protein. Most particularly, the signal peptide is the signal peptide of the CipC scaffolding protein of C. cellulolyticum, or the signal peptide of the CipA scaffolding protein of C. acetobutylicum.

"In particular embodiments, micro-organisms are provided wherein the polypeptide sequence comprises at least one carbohydrate binding module which is a carbohydrate binding module of type-3 a (CBM3a).

"In particular embodiments, micro-organisms are provided wherein the polypeptide sequence comprises at least one X module which is the X2 module of the CipC scaffolding protein of C. cellulolyticum, or the X2 module of the CipA scaffolding protein of C. acetobutylicum.

"More particularly, host cells provided according to the present invention are gram-positive bacteria, more particularly members of the class Clostridia. In further particular embodiments, micro-organisms according to the invention are micro-organisms from a Clostridium strain selected from the group comprising C. acetobutylicum and C. beijerinckii.

"Micro-organisms according to the invention may comprise one or more nucleic acids, wherein each nucleic acid comprises a sequence encoding one or more polypeptides of interest.

"In still another aspect, the invention provides a fusion protein encoded by the polynucleic acid of the invention. In addition the invention also provides a fusion protein which is fused to the signal peptide as defined herein.

"The present invention further relates to a method for the production and secretion by a host cell, more particularly a bacterial host cell, even more particularly a Clostridium host cell, most particularly a non-cellulolytic Clostridium host cell, of at least one heterologous or homologous polypeptide of interest in a biologically active form comprising introducing into said host cell of a polynucleic acid or a vector according to the invention under conditions effective to cause expression of the encoded fusion protein, wherein the encoded fusion protein is secreted by the host cell into the environment of said host cell. During secretion the signal peptide is optionally cleaved from the fusion protein. Optionally, the polypeptide of interest is simultaneously or additionally cleaved from the carrier domain.

"Accordingly, in particular embodiments, the invention provides, methods for the production and secretion by a recombinant micro-organism of at least one heterologous or homologous polypeptide of interest comprising introducing into the micro-organism a polynucleic acid encoding a fusion protein consisting of a polypeptide sequence which comprises, more particularly in this order (1) at least one signal peptide; (2) a carrier domain comprising at least one carbohydrate binding module (CBM) of the type of a cellulosomal scaffolding protein, fused to at least one X module of a cellulosomal scaffolding protein; (3) at least one polypeptide of interest; and (4) at least one peptide linker for linking the carrier domain to the polypeptide of interest, under conditions effective to cause expression of the encoded fusion protein, wherein the encoded fusion protein is secreted by the recombinant micro-organism into the environment of the recombinant micro-organism.

"A further aspect of the invention encompasses the use of a polynucleic acid, a vector or a host cell according to the invention for the production and secretion of a polypeptide of interest in a biologically active form.

"In particular embodiments of the different aspects of the invention, the polypeptides of interest comprise an enzyme such as a plant cell wall degrading enzyme, and preferably a cellulase. Most particularly, the enzyme is a cellulase of C. cellulolyticum, such as Cel48F or Cel9G. Additionally or alternatively, the polypeptide of interest comprises a cellulase CelH of S. degradans strain 2-40.

"In another embodiment, polypeptides of interest according to the invention may comprise a therapeutic protein. Such a therapeutic protein can be but is not limited to a protein selected from the group comprising therapeutic enzymes, cytokines, and antibodies, and preferably cytokines such as IL-2 or TNF.alpha.

"In yet another aspect, the invention relates to a pharmaceutical composition for the treatment of cancer comprising one of a polynucleic acid, a fusion protein, a vector, or a host cell according to the invention and at least one pharmaceutically acceptable carrier. More particularly, the pharmaceutical composition comprises a host cell, most particularly a Clostridium host cell, expressing the polynucleic acid according to the invention

"The invention further relates to a polynucleic acid, a fusion protein, a vector, or a host cell according to the invention for use as a medicament.

"In addition, the invention is directed to a polynucleic acid, a fusion protein, a vector, or a host cell according to the invention for treating cancer.

"In a further aspect, the invention provides methods of treating cancer in a subject in need thereof comprising administering a polynucleic acid, a vector, a host cell or a pharmaceutical composition according to the invention to said subject, and preferably comprising injecting said polynucleic acid, a vector, a host cell or a pharmaceutical composition at a tumor site in said subject. More particularly, the invention provides a method of treating cancer in a subject in need thereof comprising administering a host cell expressing the polynucleic acid according to the invention to said subject. Optionally, said host cell is injected at the tumor site in said subject.

"Additional aspects of the present invention will be apparent in view of the detailed description, which follows.

DESCRIPTION OF THE FIGURES

"FIG. 1 is a schematic representation of different constructs according to particular embodiments of the invention. These constructs comprise a polypeptide of interest (cellulase Cel48F or Cel9G) fused to a carrier domain and a signal peptide. The carrier domain comprises a carbohydrate binding module (CBM3a) from a cellulosomal scaffolding protein fused to one or two hydrophilic domains (Xc or Xa) originating from a same or different cellulosomal scaffolding protein(s). FIG. 1 further indicates the secretion of these constructs by C. acetobutylicum.

"FIG. 2 is a schematic representation of different constructs according to particular embodiments of the invention. The constructs comprise a polypeptide of interest (cellulase 'Cel5H') fused to a carrier domain and a signal sequence. The carrier domain comprises a carbohydrate binding module (CBM3a) from a cellulosomal scaffolding protein fused to one or two X modules (Xa). Cellulase Cel5H comprises a glycoside hydrolase family 5 domain ('5'), a polyserine linker ('sss'), a carbohydrate-binding module family 6 domain ('6'), a glutamic acid-proline-rich region ('eppv') and a C-terminal domain identified by the present inventors as a putative carbohydrate-binding module ('DZ').

"FIG. 3 demonstrates the secretion of wild-type Cel5H and Cel5H fused to a carrier domain, compared to a control strain. The carrier domain encompasses a carbohydrate binding module (CBM3a) from a cellulosomal scaffolding protein fused to two hydrophilic domains (Xa). The activity of the culture supernatant was measured on the soluble substrate para-nitrophenyl-cellobiose.

"FIG. 4 demonstrates the activity of different proteins including the fusion proteins according to particular embodiments of the invention on cellulose: activity of proteins comprising Cel9G on crystalline cellulose Avicel compared to wild-type Cel9G. The legend is as in FIG. 1.

"FIG. 5 demonstrates the activity of different proteins including the fusion proteins according to particular embodiments of the invention on different celluloic substrates; (a) activity of proteins comprising Cel5H on soluble substrate para-nitrophenyl-cellobiose; wild-type Cel5H (full line), fusion with one X module (CBM-Xa-5H; dotted line), fusion protein with two X modules (CBM-Xa-Xa-5H, dashed line) (b) activity of proteins comprising cel5H on crystalline cellulose Avicel."

For more information, see this patent application: FIEROBE, Henri-Pierre; Mingardon, Florence; Chanal-Vial, Angelique. Constructs and Methods for the Production and Secretion of Polypeptides. Filed April 9, 2014 and posted August 14, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2970&p=60&f=G&l=50&d=PG01&S1=20140807.PD.&OS=PD/20140807&RS=PD/20140807

Keywords for this news article include: Patents, Peptides, Proteins, Cell Wall, Amino Acids, Cellulosomes, Cellular Structures, Enzymes and Coenzymes, Gram-Positive Bacteria, Cell Surface Extensions, Clostridium cellulolyticum, Gram-Positive Endospore-Forming Rods, Gram-Positive Endospore-Forming Bacteria.

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