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SM-protein based secretion engineering

A protein and heterologous protein technology, applied in genetic engineering, peptide/protein components, animal/human proteins, etc., can solve problems such as limited understanding of basic regulatory mechanisms, and achieve the effect of reducing costs and improving protein yields

Inactive Publication Date: 2010-12-01
BOEHRINGER INGELHEIM PHARM KG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently two major obstacles on the road to targeted manipulation of the secretory transport machinery: the still limited understanding of the underlying regulatory mechanisms and the challenge of preventing the bottleneck from moving far downstream during secretion

Method used

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  • SM-protein based secretion engineering
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] Example 1: Localization of Sly1 and Munc18c in HEK-293 along the secretory pathway.

[0278] We used RT-PCR based analysis to characterize the expression of SM proteins Sly1 and Munc18 isoforms (a, b, c) in HEK-293. as in figure 1 As shown in a and 1b, sly1 (NM_016160) and munc18c (NM_007269) were expressed at high levels, muc18b (NM_006949) was expressed in a small amount, and the transcript of neuron-specific munc18a (NM_003165) could not be detected. By Western blotting ( figure 1 c) To confirm the SM protein profile. Sly1 and Munc18 were analyzed by co-expression of YFP-Sly1 (pRP32) and CFP-syntaxin 5 (pRP40) or YFP-Munc18c (pRP23) and CFP-syntaxin 4 (pRP29) in HEK-293. Intracellular localization of I-type Munc18c. Syntaxin 5 is a SNARE that binds to Sly1 localized in the Golgi apparatus, and syntaxin 4 is a SNARE that interacts with Munc18c bound to the cell membrane. Confocal microscopy showed that Sly1 showed extremely dense perinuclear co-localization wit...

Embodiment 2

[0279] Example 2: Sly1 and Munc18 regulate protein secretion.

[0280] SM proteins are known to control vesicle fusion necessary for intracellular protein transport, but their role for protein secretion remains unknown. To characterize the effect of Sly1 and Munc18 on overall exocytosis, we designed shRNAs specific to these SM proteins. Knockdown of Sly1 and Munc18c was demonstrated by the following method: cells were encoded with bicistronic Sly1 (pRP3; P hCMV -sly1-IRES-eGFP-pA) and encoding Munc18c (pRP4; P hCMV -munc 18c-IRES-eGFP-pA) reporter construct was co-transfected with specific and non-specific control shRNA, and fluorescent microscopy was performed ( figure 2 ). The ability of each shRNA to knock down endogenous Sly1 and Munc18c expression by up to 70% was demonstrated in HEK-293 ( image 3 a and 3c). To analyze the effect of Sly1 and Munc18c knockdown on the overall protein secretion ability of mammalian cells, we combined pSEAP2 control and pRP5 (shRNA sly...

Embodiment 3

[0281] Example 3: Ectopic expression of Sly1 and Munc18c improves the secretory ability of mammalian cells.

[0282] Ectopic expression of Sly1 or Munc18c in CHO-K1 ( Figure 4 a After 4b, 4c), SEAP, SAMY or VEGF 121 Heterologous production was increased up to 5-fold, which is consistent with the promoter used to initiate transcription of the product gene (P SV40 ,P hCMV ,P EF1α ) is irrelevant. Similar results were also observed when using HEK-293 cells (data not shown). Because the mRNA levels of SEAP, SAMY, and VEGF were essentially constant with or without elevated Sly1, Munc18c, or both ( Figure 4 d), so the increase in heterologous protein production is mediated by post-translational mechanisms. Our results are consistent with previous studies arguing that the Munc18 protein inhibits exocytosis in a range of cell types, including adipocytes and muscle cells (Riento et al., 2000; Kanda et al., 2005; Tellam et al., 1997; Thurmond et al. et al., 1998), which is the ...

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Abstract

The present invention concerns the field of cell culture technology. It describes a novel method for enhancing the secretory transport of proteins in eukaryotic cells by heterologous expression of Munc18c, Sly1 or other members of the SM protein family. This method is particularly useful for the generation of optimized host cell systems with enhanced production capacity for the expression and manufacture of recombinant protein products.

Description

technical field [0001] The present invention relates to the field of cell culture technology. It relates to a method of producing proteins and a method of generating new expression vectors and host cells for biopharmaceutical production. The invention additionally relates to pharmaceutical compositions and methods of treatment. Background technique [0002] The market for biopharmaceuticals for human therapy continues to grow rapidly, with 270 novel biopharmaceuticals being evaluated in clinical studies and an estimated $30 billion sold in 2003. Biopharmaceuticals can be manufactured from a variety of host cell systems including bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells, including cell lines derived from humans. Currently, an increasing number of biopharmaceuticals are manufactured from eukaryotic cells due to their ability to properly process and modify human proteins. Thus, successful and high-yield production of biopharmaceuticals fro...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N15/11C12N15/12C12N15/63A61K38/00A61K39/00A61K48/00C12N1/15C12N1/21C12N5/10C12N15/113
CPCC12N2310/14C12P21/02C12N15/113C07K14/705C12N2310/11A61P29/00A61P35/00A61P37/00A61P37/02
Inventor 希托·考夫曼埃里克·贝克劳尔·弗洛林马丁·弗森尼格彭仁旺乔伊·M·斯图茨
Owner BOEHRINGER INGELHEIM PHARM KG
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