Login

Forgot your password?
New User?
Remember me
banner banner
 
United States Biological
August 2009 Issue 1| Subscribe www.usbio.net
Newsletter

Human Cytokines :
human cells   Recombinant human cytokines are essential for the expansion and differentiation of human stem cells. As stem cell research continues to advance and findings are translated to the clinic, there is a growing need for recombinant cytokines to support the clinical development of cellular therapies. United States Biological is supplying an efficient human-cell based technology for the scalable production of human cytokines. An expanding range of authentic, tag-free cytokines, is available, including difficult-to-express protein members of the TGF-beta superfamily. United States Biological's authentic cytokines can be used as cost effective, highly preferred reagents for stem cell research and clinical development.

InterleukinInterleukin  

Some of USBio's Recombinant Human Interleukins:

Recombinant Human Interleukin 1 beta
IL-1beta expressed in human 293 cells has a very high activity with ED50 of 34pg/ml. The cytokine is more than 95% pure. In comparison with the E. Coli expressed protein, IL-1beta is 50% more potent to promote human CD4+ T cells differentiation into Th17 cells.

Recombinant Human Interleukin 2
IL-2 expressed in human 293 cells is a glycosylated monomer with a molecular mass of
15kDa. Production in human 293 cells offers
authentic glycosylation; which contributes to stability in cell growth media and other
applications.

Recombinant Human Interleukin 4
IL-4 has 5-fold higher stability as compared to the E. coli expressed product. It has a demonstrated greater stability under cell culture conditions making it ideal for efficient generation of Human Dendritic Cells.

Recombinant Human Interleukin 10
IL-10 is expressed in human 293 cells as a glycosylated homodimer with an apparent molecular mass of 18kD. Glycosylation contributes to stability in cell growth media and other applications. The protein is highly active with ED50 of 0.3-1.5ng/ml. The cytokine is more than 95% pure. IL-10 is predominantly a noncovalent homodimer 35~40kD at neutral pH.


USBio's 2009 catalog is ready for shipping.
US Bio CatalogIf you haven't reserved your copy yet, you can do it now. It features an expanded reference section and over 20,000 new products.


This month's Special:
10% off Biologically Relevant Noggin, N3100-05E

Noggin
Expression of pluripotent stem cell markers of hES colonies cultured in the presence of Noggin (N3100-05E,10-20pg/ml) expressed in human cells. A. Green TRA1-60 B. Green SSEA-4 C. Green TRA1-81 D. OCT 3/4 (Blue=Nuclear)

Noggin is a secreted homodimeric glycoprotein that is an antagonist of bone morphogenetic proteins (BMPs). During culture of human embryonic stem (hES) cells without feeder layer or conditioned medium (but with addition of FGF basic), the addition of Noggin allows the stem cells to maintain their undifferentiated, pluripotent state. Commercially available Noggin products are produced in a variety of forms none of which are authentic: non-glycosylated protein expressed in E. coli; glycosylated Fc-fusion protein expresed in NS0, for example. USBiological supplies Noggin in a stable, engineered human 293 cell expression system. The protein is expressed as an authentic glycosylated, disulfide-linked dimer.


Human Cell Expression Cytokines
Functional Activities

Functional activities of human proteins depend on proper folding, phosphorylation, disulfide bridge formation, and proteolytic processing or appropriate glycosylation. Ideally, recombinant human proteins produced in human cells are more authentic in terms of both physical properties and biochemical functions. However, the current process of human cell expression requires a large quantity of DNA and medium supplemented with bovine serum. Hence, it is often a daunting task to produce sufficient amount of high quality proteins for drug discovery or diagnostic development (1-100mg) at acceptable cost. United States Biological is now supplying a high yield, cost-effective and versatile system to produce active recombinant human proteins in a human 293-cell expression system to meet the special needs of the broad scientific community and drug discovery industry. In this system, we have broken key bottleneck steps to increase protein production and reduce cost, including

  1. Engineered human cell line adapted to suspension for growth in serum-free and chemically defined medium

  2. Proprietary expression vectors driven by strong promoters and signal peptides

  3. Large scale transient expression with small quantity of DNA

  4. Rapid creation of stable human cell lines and adaptation to serum-free medium

  5. Short time to scale up production

  6. Efficient tag or tag-free purification

  Using this expression system, we have produced a large number of highly active human cytokines, kinases and phosphatases. Our technology is particularly suitable for the production of high-authenticity human proteins in medium scale for research and diagnostic reagents, drug screening targets, and therapeutic proteins for preclinical evaluation. Key differences of recombinant human protein production in different systems are illustrated below:

Expression system E. coli Insect cell CHO cell Human cell
Protein folding + ++ +++ ++++
Phosphorylation - ++ +++ ++++
Proteolytic processing - + +++ ++++
Glycosylation - Poor Not human-like Authentic

  Cytokines are a group of proteins and polypeptides that organisms use as signaling molecules. Most cytokines are glycoproteins less than 30kD in size and bind to specific, high-affinity cell surface receptors. Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory and infectious diseases and widely used in research, diagnostics and therapeutics. Currently, these proteins are predominantly produced in non-human cells (e.g. E. coli, SF9, CHO) and therefore lack authenticity due to the absence of physiologically relevant glycosylation. In addition, a number of important cytokines are not commercially available due to inadequate proteolytic processing, protein folding or other post-translational modifications that occur in the non-human cell expression systems. United States Biological is now supplying recombinant cytokines based on human cell expression technology, for the scalable production of human cytokines. These authentic cytokines are preferred reagents for stem cell, cancer, and inflammation research, and also antibody development.

  Cytokines produced in E. coli are not glycosylated and may expose cryptic or normally hidden epitopes. Similarly, cytokines produced in SF9 or CHO cells have post-translational modifications that are not human-like. Because of these factors, antibodies may have different affinities depending on whether they were created from human cell expressed protein antigens or non-human cell expressed protein antigens. Indeed, Western blot analysis shows that monoclonal antibodies raised against a full-length protein from non-human cells poorly recognize the recombinant human cytokines produced in human cells using this human cell expression technology. In contrast, these antibodies are highly reactive to the proteins that may correspond to micro-aggregates. These results indicate that recombinant cytokines from a human cell expression system are highly preferred antigens to raise antibodies as well as use as standards in ELISA assays.

IL-23 (I8443-40G, Interleukin 23, Recombinant, Human (IL-23) (Human 293 Cells))

   Currently, commercially available recombinant IL-23 cytokine is produced as a heterodimeric or fusion protein from an insect cell expresion system. United States Biological has IL-23 available that is produced in a stable cell culture of engineered human HEK293 cells. The protein is expressed as a disulfide-linked dimer of 55kD and, due to the scalability of the stable culture, can be cost-effectively produced. (Fig. 1).
IL23 width=

Figure 1. Highly purified glycosylated, dimeric recombinant human IL-23 (I8443-40G) expressed in an HEK293 human cell expression system. Lane 1: MW standards, Lane 2: non-reducing conditions, Lane 3: reducing conditions.

IL-17-producing CD4+ T cells (Th17 cells) have been identified as a unique subset of T helper cells that develop along a pathway that is distinct from the Th1 and Th2-cell differentiation pathways. This finding has provided exciting new insights into immunoregulation, host defense and the pathogenesis of autoimmune diseases. Recently it has been shown that TGF-s1 , IL-1s, IL-6 and IL-23 are important in driving human Th17 differentiation. The bioactivities of IL-23 from human and insect cells were first determined by the dose-dependent secretion of IL-17 from mouse splenocytes activated with 10 ng/ml PMA, which shows that IL-23 (Human 293 Cells) is ten fold more active. (Fig. 2).

IL23 width=

Figure 2. IL-23 (I8443-40G) activities determined by the dose-dependent secretion of IL-17 from mouse splenocytes activated with 10ng/ml PMA.

The activities were further assayed with human CD4+ cells isolated from a healthy donor and stimulated with 10ug/ml plate bound anti-CD3 and 10ug/ml soluble anti-CD28 in the presence of Th17 polarizing cytokines. After 5 days, the supernatants were harvested and IL-17 was measured by ELISA. The results show that I8443-40G is 100-fold more potent for inducing IL-17 secretion in two independent studies, maximum induction was achieved with 0.1 ng/ml I8443-40G vs. 10ng/ml with insect cell-produced IL-23 (Fig. 3). These results demonstrate that authenic human tcell expressed cytokines can induce Th17 cell differentiation at physiologically relevant concentration and may lead to more accurate scientific understanding of human biological process.

IL23 width= Figure 3. IL-23 (I8443-40G) activities determined by the dose-dependent secretion of IL-17 from human CD4+ T cells stimulated with 2mg/ml plate bound anti-CD3 and 1mg/ml soluble anti-CD28 in the presence of Th17 polarizing cytokines.

A rapidly expanding range of human 293 cell expressed cytokines are available from United States Biological. The proteins are manufactured to high quality standards and provide high biological activity, lot-to-lot consistency and low endotoxin levels.

Recombinant Cytokines Expressed in Human 293 Cells

A2281-45A Androgen Receptor Coactivator ARA55, Recombinant, Human ( Transforming Growth Factor beta1-induced Transcript 1, TGFB1I1) (Human 293 Cells)
B2553-08B Bone Morphogenetic Protein 2, Recombinant, Human (BMP-2) (Human 293 Cells)
B2553-20C Bone Morphogenic Protein 4, Recombinant, Human (BMP4, DVR4) (Human 293 Cells)
C2448-02Q CD117, Recombinant, Human (c-kit, Stem Cell Factor, SCF Receptor, Steel Factor Receptor, Mast Cell Growth Factor, MGF) (Human 293 Cells)
E3455-07B Erythropoietin, Recombinant, Human (EPO) (Human 293 Cells)
F4211-51 Fibroblast Growth Factor 8b, Recombinant, Human (FGF8b) (Human 293 Cells)
F4600-10C Flt3 Ligand, Recombinant, Human (Fms-like Tyrosine Kinase 3 Ligand, flk2 Ligand) (Human 293 Cells)
G8950-02C Granulocyte Colony Stimulating Factor, Recombinant, Human (G-CSF) (Human 293 Cells)
G9000-07A Growth Hormone, Recombinant, Human (hGH) (Human 293 Cells)
H2005-02A Hepatocyte Growth Factor, Recombinant, Human (HGF, Scatter Factor, SF, Hepatopoietin A) (Human 293 Cells)
I0945-06A Inhibin A, beta, Recombinant, Human (Activin) (Human 293 Cells)
I7661-64D Interferon alpha 2b, Recombinant, Human (IFNa2b) (Human 293 Cells)
I7662-06C Interferon alpha A (alpha 2a), Recombinant, Human (IFNaA, IFNa2a) (Human 293 Cells)
I7663-07A1 Interleukin 1 beta, Recombinant, Human (IL-1b) (Human 293 Cells)
I7663-26A4 Interleukin 2, Recombinant, Human (IL-2) (Human 293 Cells)
I7663-37E Interleukin 3, Recombinant, Human (IL-3) (Human 293 Cells)
I8426-04D Interleukin 4, Recombinant, Human (IL-4, B Cell Stimulatory Factor 1, BSF1, Binetrakin, HCGF, Hodgkin's Cell Growth Factor, IA Inducing Factor, Lymphocyte Stimulatory Factor 1, Macrophage Fusion Factor, Mast Cell Growth Factor 2, MCGF2, MFF, MGC79402, Pitrakinra, T Cell Growth Factor 2, TCGF2) (Human 293 Cells)
I8428-02B4 Interleukin 6, Recombinant, Human (IL-6, B cell Stimulatory Factor 2, BSF2, Cytotoxic T cell Differentiation Factor, CTL Differentiation Factor, CDF, Hepatocyte Stimulating Factor, HSF, Hybridoma Growth Factor, HGF, Hybridoma Plasmacytoma Growth Factor, HPGF, Interferon beta 2, IFNB2) (Human 293 Cells)
I8432-13P Interleukin 10, Recombinant, Human (IL-10, Cytokine Synthesis Inhibitory Factor, CSIF) (Human 293 Cells)
I8434-01B Interleukin 12, Recombinant, Human (IL-12, Cytotoxic Lymphocyte Maturation Factor 1, CTL Maturation Factor, TcMF, CLMF p35, Natural Killer Cell Stimulatory Factor 1, NFSK1, p35, T cell Stimulating Factor, TSF) (Human 293 Cells)
I8439-10D Interleukin 17, Recombinant, Human (IL-17, CTLA8, IL-17A, Interleukin-17A precursor, Cytotoxic T-lymphocyte-associated antigen 8) (Human 293 Cells)
I8439-28B Interleukin 17F, Recombinant, Human (IL-17F) (Human 293 Cells)
I8443-40G Interleukin 23, Recombinant, Human (IL-23)
K0201-01C Keratinocyte Growth Factor, Recombinant Human (KGF, Fibroblast Growth Factor 7, FGF-7) (Human 293 Cells)
L1662-94 Lefty B, Recombinant, Human (Left-Right Determination, Factor B, LEFTB, LEFTY1) (Human 293 Cells)
M1201-01D Macrophage Colony Stimulating Factor, Recombinant, Human (M-CSF, CSF-1, Lanimostim, Macrophage colony-stimulating factor 1 precursor, MCSF, MGC31930) (Human 293 Cells)
N3100-05E Noggin, Recombinant, Human (Human 293 Cells)
O6500-05A Oncostatin M, Recombinant, Human (OSM) (Human 293 Cells)
T8250-26A Transforming Growth Factor beta2 Receptor, Recombinant, Human (TGFb2R) (Human 293 Cells)
T8250-45E Transforming Growth Factor beta 3, Recombinant, Human (TGFb3) (Human 293 Cells)
T9160-02S Tumor Necrosis Factor alpha, Recombinant, Human (TNFa) (Human 293 Cells)
V2110-04H Vascular Endothelial Growth Factor, Recombinant, Human (VEGF) (Human 293 Cells)
V2110-15C Vascular Endothelial Growth Factor 165, Recombinant, Human (VEGF165) (Human 293 Cells)