COmpositions and methods for high-level, large-scale production of recombinant proteins

A technology of recombinant protein and assembly, which is applied in the field of overexpression of recombinant protein assembly, bidirectional UCOE vector, high-level large-scale production of polypeptide, and UCOE-based vector, which can solve problems such as intergenic transcription interference

Inactive Publication Date: 2004-09-29
ML LAB PLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, traditional methods for expressing antibody heavy and light chains require co-transfection of plasmids with heavy and light chain coding regions, respectively, but this is still diff

Method used

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  • COmpositions and methods for high-level, large-scale production of recombinant proteins
  • COmpositions and methods for high-level, large-scale production of recombinant proteins
  • COmpositions and methods for high-level, large-scale production of recombinant proteins

Examples

Experimental program
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Effect test

preparation example Construction

[0078] Preparation and selection of expression vectors

[0079] According to the present invention, vector systems suitable for expression of recombinant proteins and / or polypeptides include one or more of the following features: (a) ease of manipulation; (b) integration site-independent elements enabling high-level expression; (c) enabling expression elements that are resistant to silencing / repression, resulting in durable, stable long-term expression; and (d) elements that are expressed at high levels in different cell types and in different species.

[0080] In order to express the protein and / or polypeptide of interest, the nucleotide sequence encoding the polypeptide or its functional equivalent is inserted into an appropriate expression vector, that is, the vector contains elements necessary for the transcription and translation of the inserted coding sequence. Methods for constructing an expression vector containing a sequence encoding a polypeptide of interest and ap...

Embodiment 1

[0170] Expression of Recombinant Antibodies Using UCOE-Based Vector Systems

[0171] This example compares the level of recombinant antibody expression using vectors with and without UCOE.

[0172] use as figure 1 The indicated vectors containing human RNP UCOE express the engineered human antibody Ab3. The same vector was also constructed without the UCOE element. In this example, the Ig heavy chain coding sequence contains the modified human V region sequence, which is located upstream of the genomic DNA fragment encoding the human Igγ-1 constant region and keeps the same reading frame. The coding region of the Ig light chain contains the modified human V region sequence, which is located upstream of the cDNA fragment encoding the human Igκ constant region and keeps the same reading frame with it. The vector expressing the Ig heavy chain also contains a neo selection marker gene, and the vector expressing the Ig light chain contains a hygromycin selection marker. refer ...

Embodiment 2

[0175] High-level, large-scale expression in CHO host cell lines transfected with a UCOE-based expression vector system

[0176] Co-transfect CHO-S cells with a vector containing the UCOE antibody expression cassette (e.g. figure 1 shown) to obtain the transformed human antibody Ab1. The Ig heavy chain coding sequence contains the modified human V region sequence, which is located upstream of the genomic DNA fragment encoding the human Igγ-4 constant region and keeps the same reading frame. The coding region of the Ig light chain contains the modified human V region sequence, which is located upstream of the cDNA fragment encoding the human Igκ constant region and keeps the same reading frame. The vector expressing the Ig heavy chain also contains a neo selection marker gene, and the vector expressing the Ig light chain contains a hygromycin selection marker. refer to Figure 2B .

[0177]Transfections were performed with lipofectamine (Life Technologies) according to the...

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Abstract

The present invention discloses compositions and methods for high-level, large-scale production of recombinant proteins. Exemplary combinations comprise one or more expression vectors capable of high-level expression of recombinant proteins and/or polypeptides, and immortalized host cell lines capable of growth in serum-free, suspension culture conditions. Bi-directional UCOE vectors allow simultaneous high-level expression of two or more recombinant proteins and/or polypeptides on a single UCOE-based plasmid vector.

Description

Background of the invention [0001] field of invention [0002] The present invention relates generally to gene expression and protein production, and more particularly to compositions and methods for overexpressing recombinant proteins. Such combinations and methods are useful in high-level, large-scale production of recombinant proteins. [0003] Related technical description [0004] A major goal of the biotechnology industry is the development of stable cell line-based systems for large-scale expression of recombinant proteins, such as recombinant antibodies. Standard methods for developing suitable recombinant host cell lines are time-consuming and laborious. Routinely, cells are cultured under conditions containing fetal bovine serum, such as CHO-K1 or CHO DUX, and the target expression vector is transfected into the cells. The entire cell population is then screened to remove cells that have not been transformed into the expression vector. All vector-containing c...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N5/06C12N5/10C12N15/11C12N15/85C12P21/02
CPCC12N2510/04C12N2830/205C12N2830/46C12N2830/42C12N2840/203C12N2800/108C12N2840/20C12N15/11C12N15/85C12N15/63C12N5/10C12N15/67
Inventor 特里什·本顿克里斯多佛·罗伯特·贝宾顿卡尔拉·安·亨宁戴维·J·金罗伯特·克龙比向少
Owner ML LAB PLC
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