Introduction of Mammalian expression systems

The mammalian protein expression system that Medicilon provides is based on a serum-free cultivated system to provide the gene expression optimization, transient expression, stable expression and antibody production and other eukaryotic protein services.

Researchers of Medicilon established a well-developed mammalian protein expression system and purification services platform, which offers the expression and purification services of a variety of recombinant protein , antibody or fragment of antibody.

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The present invention features mammalian expression systems with improved production yields, and method of using these systems to produce desired proteins. In one embodiment, the expression systems of the present invention comprise genetically-engineered mammalian host cells cultured in a medium that contains an effective amount of heparin or heparin-like molecules. The presence of heparin or heparin-like molecules significantly increases protein production by the cultured cells. The present invention also features the use of constitutively-active components of FGFR-I -mediated signal transduction pathways to improve protein production by cultured mammalian cells. Co-expression of such a component with a protein of interest markedly increases the production yield of the protein of interest.

With recent advances in genomics and proteomics, the ability to clone and express recombinant proteins in large amounts has become increasingly important. The ability to purify high levels of proteins is important in the human pharmaceutics and biotechnology setting, for production of protein pharmaceuticals such as insulin, as well as in the basic research setting, for example to crystallize a protein to allow determination of its three-dimensional structure. Proteins that are otherwise difficult to obtain in quantity can be overexpressed in a host cell and subsequently isolated and purified.

Bacterial expression systems have been one approach to expression and purification of recombinant proteins. However, expression of many eukaryotic polypeptides, and particularly mammalian proteins, in bacterial cells has frequently produced disappointing and unsatisfactory results because conditions and the environment in the host cells were not conducive to correct folding and modification of the eukaryotic protein.

Yeast expression systems offer certain advantages for the production of some eukaryotic proteins, because they have secretory pathways and have the ability to perform some limited post-translational modifications. However, yeast systems often lead to improper folding of disulfide linked proteins, and may result in hypoglycosylation.

The use of mammalian cells for the production of proteins offers the important advantages of providing correct protein folding as well as the appropriate post-translational modifications, such as glycosylation. However, many mammalian expression systems do not produce large quantities of desired proteins.

The present invention features the use of heparin, heparin-like molecules, or fibroblast growth factor receptor (FGFR) agonists to increase protein production by mammalian host cells. The present invention also features the use of constitutively-active FGFRs or their downstream effectors to stimulate protein production by mammalian host cells.

In one aspect, the present invention provides mammalian expression systems with improved protein production yields. These expression systems include genetically-engineered mammalian cells cultured in a medium that contains an effective amount of heparin or heparan sulfate glycosaminoglycans. Each of the genetically-engineered host cells includes a recombinant expression cassette encoding a protein of interest. The presence of heparin or heparan sulfate glycosaminoglycans in the culture medium significantly increases the yield of the protein of interest.The amount of heparin or heparan sulfate glycosaminoglycans used in the present invention can be any amount that is effective for promoting protein production by the cultured host cells. In one embodiment, a culture medium employed in the present invention includes from about 1 to about 1,000 μg/ml of heparin or heparan sulfate glycosaminoglycans. In another embodiment, a culture medium employed in the present invention includes from about 10 to about 200 μg/ml of heparin or heparan sulfate glycosaminoglycans .In yet another embodiment, a culture medium employed in the present invention is a serum-free medium which includes an effective amount of fibroblast growth factor 2 or other FGFs, in combination with heparin or heparan sulfate glycosaminoglycans, for increasing protein production by the cultured host cells. In many examples, the culture medium includes, without limitation, from about 10 to about 500 ng/ml of FGF-2.In another aspect, the mammalian expression systems of the present invention include genetically-engineered mammalian cells cultured in a medium that contains an effective amount of an FGFR-I activation agent. Each of these genetically-engineered cells includes a recombinant expression cassette encoding a protein of interest. The presence of the FGFR-I activation agent in the culture medium markedly increases the yield of the protein of interest. Examples of FGFR- 1 activation agents suitable for the present invention include, but are not limited to, FGFs, heparins, heparan sulfate glycosaminoglycans, or other heparin-like molecules. Agents capable of activating other FGFRs can also be used. In one embodiment, the FGFR-I activation agent employed in the present invention includes both heparin or heparan sulfate glycosaminoglycans and FGF-2.In still another aspect, the mammalian expression systems of the present invention include genetically-engineered mammalian cells, each of which includes one or more recombinant expression cassettes that encode a protein of interest and a constitutively-active component of an FGFR-I -mediated signal transduction pathway. In one example, the constitutively-active component of the FGFR-I- mediated signal transduction pathway is a constitutively-active FGFR-I protein.The present invention also features the use of β-xylosides or other glycosaminoglycan biosynthesis inducers to improve protein production by mammalian cells. Non-limiting examples of β-xylosides suitable for this purpose include 4-methylumbelliferyl-β-D-xyloside, p-nitrophenyl-β-D-xyloside, and benzyl-β-D-xyloside. In one example, mammalian host cells are cultured in a medium that comprises from about 50 or about 100 μg/ml of 4-methylumbelliferyl- β-D-xyloside. The use of β-xylosides significantly increases the protein production yield of mammalian host cells.

Any protein of interest can be produced using the mammalian expression systems of the present invention. Non-limiting examples of these proteins include insulins, growth hormones, growth factors, erythropoietin proteins, follicle- stimulating hormones, interferons, interleukins, cytokines, colony stimulating factors, coagulation factors, tissue plasminogen activators, parathyroid hormones, bone morphogenetic proteins, keratinocyte growth factors, granulocyte colony- stimulating factors, granulocyte-macrophage colony-stimulating factors, glucagons, thrombins, thrombopoietins, protein C, secreted frizzled-related proteins, selectins, antibodies, or viral proteins. These proteins can be secreted, cytosolic, or membrane-bound proteins. They can be used for therapeutic, prophylactic, diagnostic, or other medical purposes. In many examples, the proteins produced by the present invention are incapable of interacting with cell-surface heparan sulfate proteoglycans to induce cellular internalization of these proteins.The present invention further features pharmaceutical compositions including proteins produced by the mammalian expression systems of the present invention.

In addition, the present invention features methods for producing desired proteins. In one aspect, the methods of the present invention include culturing mammalian host cells in a medium, wherein each of the host cells includes a recombinant expression cassette encoding a protein of interest, and the culture medium contains an effective amount of heparin, heparan sulfate glycosaminoglycans, or an FGFR-I activation agent for increasing the production of the protein of interest by the host cells; and isolating the protein of interest from the host cells or the culture medium.In one embodiment, the recombinant expression cassette is carried by an expression vector which is transiently introduced into the host cells. The heparin or heparan sulfate glycosaminoglycans are added to the culture medium at least 24 hours after the expression vector is transiently introduced into the host cells. In another embodiment, the heparin or heparan sulfate glycosaminoglycans are added to the culture medium at least 48 hours after the expression vector is transiently introduced into the host cells.