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Mammalian expression systems

An expression system, mammalian technology, applied in the direction of digestive system, endocrine system diseases, animal/human proteins, etc., can solve the problem of not being able to produce a large amount of desired protein

Inactive Publication Date: 2008-04-09
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many mammalian expression systems do not produce large quantities of the desired protein

Method used

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  • Mammalian expression systems
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Examples

Experimental program
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example 1

[0091] Example 1. Cell Culture and DNA Constructs

[0092] Make mammalian cell lines (HEK293-FT, HEK293-EBNA, CHO-DUKX and Lec.3.2.8.1) in 5% CO 2 Grow and maintain at 37°C in a humidified incubator. HEK293 cells were cultured in dendritic 293 medium (Invitrogen) supplemented with 5% fetal bovine serum (FBS). The CHO-DUKX stable cell line was grown in alpha medium containing 10% FBS and 200 nM methotrexate (MTX). HEK293 stable cell lines were cultured in alpha medium containing 10% FBS and 100 nM MTX.

[0093] Perform transient expression in a 50 mL rotator or a 1 L rotator. For a 50-ml culture volume, 25 μg of plasmid DNA was mixed with 400 μg of polyethyleneimine (PEI, 25 kDa, linear, neutralized with HCl to pH 7.0, 1 mg / ml (Polysciences, Warrington, PA)) in 2.5 ml without Mix in serum-containing 293 medium. For 1 L culture volume, mix 500 µg DNA with 4 mg PEI in 50 mL serum-free 293 medium. The mixture was then mixed with 50 mL or 1 L of HEK293 cells at 0.5 x 10 6 Cell...

example 2

[0096] Example 2. Heparin enhances the production of sFRP-1 by transfected cells

[0097] The C-terminal his6-tagged sFRP-1 was transiently expressed in HEK293-FT cells as described in Example 1. 50 μg / ml heparin (Sigma Chemical) was added to the cell culture medium during or after DNA transfection ("post-transfection induction time", shown in Figure 2). Conditioned medium was harvested at different time points ("growth time", shown in Figure 2). Protein samples were separated by SDS-PAGE and subjected to Western blot analysis with mouse monoclonal anti-his4 antibody (Qiagen) at 0.2 μg / ml (Figure 2). Western blotting was performed according to Zhong et al. (2004) FEBS Lett.562: Implementation as described in 111-117. As shown in Figure 2, heparin greatly increased sFRP-1 production by transfected HEK293 cells. The greatest increase was observed when heparin was added to the medium 48 hours after DNA transfection. In other experiments, this increase was not observed with re...

example 3

[0106] Example 3. FGF-2 Enhances Protein Production by Transfected Cells

[0107] Cells from the HEK293 cell line stably overexpressing sFRP-1 (clone 100-5) were grown to confluence in 6-well plates; the serum-free medium was then replaced with fresh serum-free medium containing 50 μg / ml heparin Medium. 50 ng / ml of fibroblast growth factor-1 (FGF-1, Sigma Chemical Company) or fibroblast growth factor-2 (FGF-2, Sigma Chemical Company) was added to the medium in the corresponding well. Conditioned medium was harvested 48 hours after medium replacement. Protein samples were separated by SDS-PAGE and subjected to immunoblot analysis with mouse monoclonal anti-his4 antibody (Figure 10). As demonstrated in Figure 10, the combination of FGF-2 and heparin significantly increased the production of sFRP-1 by stably transfected HEK293 cells.

[0108] Thus, FGF-2 and heparin can regulate protein synthesis in a post-transcriptional manner, as Northern blot analysis showed that sFRP-1 mR...

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Abstract

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.

Description

technical field [0001] The present invention relates to mammalian expression systems and methods of using said mammalian expression systems to produce desired proteins. Background technique [0002] Recently, with advances in genomics and proteomics, the ability to clone and express recombinant proteins in large quantities has become increasingly important. The ability to purify high levels of protein is important in human pharmaceutical and biotechnology settings for the production of protein medicines such as insulin, as well as in basic research settings to, for example, crystallize proteins to determine their three-dimensional structure. Also proteins that are difficult to obtain in large quantities may be overexpressed in host cells and subsequently isolated and purified. [0003] Bacterial expression system has become a method for recombinant protein expression and purification. However, expression of many eukaryotic polypeptides and (especially) mammalian proteins i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C08B37/10C07K14/71
CPCC12P21/02A61P1/04A61P3/10A61P5/10A61P5/18A61P5/30A61P7/00A61P7/04A61P17/00A61P19/00A61P37/04
Inventor 钟晓天罗纳德·克里茨马克·施塔尔
Owner WYETH LLC
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