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Fusion peptides for enhancing protein expression

A technology of fusion peptide and expression cassette, applied in the field of expression cassette, can solve the problems of low expression rate, high cost, low yield of recombinant protein, etc.

Inactive Publication Date: 2015-11-11
GLYCOTOPE GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many expression systems using higher eukaryotic cell lines suffer from rather low expression rates of the desired protein, resulting in low yields and high costs of recombinant proteins

Method used

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  • Fusion peptides for enhancing protein expression
  • Fusion peptides for enhancing protein expression
  • Fusion peptides for enhancing protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Comparison of Erythropoietin Expression Alone or as a Fusion Protein

[0112]In this assay, the effect of fusion to a portion of the extracellular domain of glycophorin A on the expression of the model protein human erythropoietin (EPO) was tested. The nucleic acid sequence encoding EPO was cloned into a standard vector for eukaryotic expression containing the EF-la promoter and the dihydrofolate reductase (DHFR) gene as a selectable marker. A leader sequence (extracellular expression signal) for IgK or GM-CSF is present for secretory expression of the EPO. In addition, a nucleic acid sequence encoding amino acids 1 to 38 of mature human glycophorin A (GYPAex.reg.) was cloned into the vector in the same reading frame, between the extracellular expression signal and the EPO or after the EPO . In the control vector, the glycophorin construct was not inserted, but only the extracellular expression signal and the EPO were encoded. The constructs used in this a...

Embodiment 2

[0122] Example 2: Comparison of Factor VII Expression Alone or as a Fusion Protein

[0123] The effect of fragments of the extracellular region of glycophorin on the production of yet another target protein, Factor VII, was also analyzed. The construct was designed and assayed as described in Example 1, except that the glycophorin extracellular domain tag consisted of amino acids 2 to 38 of mature human glycophorin A. The leader sequence of Factor VII was used as signal peptide. A hemoglobin tag (HB-tag) was fused to the N-terminus of Factor VII as a further control. Use the following constructs:

[0124] Control 1: Leader Sequence - Factor VII

[0125] Control 2: Leader-HB-Tag-Factor VII

[0126] ETag2.1: Leader Sequence-GYPAex.reg.-Factor VII

[0127] ETag2.2: Leader-Factor VII-GYPAex.reg.

[0128] The average production rates obtained in this assay are listed in the table below:

[0129] Table 2

[0130]

[0131]

[0132] These data again demonstrate that the ...

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Abstract

The present invention pertains to the field of recombinant protein production. Novel peptides derived from the extracellular region of a glycophorin protein are provided which enhance the expression rate of proteins or peptides of interest when expressed as fusion protein together with said novel peptides.

Description

field of invention [0001] The present invention relates to novel peptides that can be used to enhance the production of proteins of interest. The peptides are derived from the extracellular domain of glycophorin and are used as part of a protein fusion to the protein of interest. The invention particularly provides expression cassettes comprising such peptides as part of the open reading frame. Background of the invention [0002] The production of recombinant proteins is a major aspect of the biotechnology industry today. This is becoming increasingly important due to the increasing number of applications in the market that require large amounts of high-quality protein. Food production and especially pharmaceuticals are two major areas where the demand for recombinant proteins is steadily increasing. Achieving a commercially viable process requires a higher production efficiency and thus lower cost of the final product. [0003] But at the same time, high product qualit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435
CPCC07K14/47C12N15/85C07K14/505C07K2319/35C07K2319/50
Inventor S·戈勒茨A·丹尼尔奇克D·雅恩
Owner GLYCOTOPE GMBH