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Vectors and methods for recombinant protein expression

A technology of expression vectors and eukaryotic expression vectors, applied in the field of vectors and methods for recombinant protein expression, can solve the problems of increased pressure, inability to select and produce high-expression cell lines, etc.

Inactive Publication Date: 2013-11-13
ADV BIOLOGICS
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Second, selection pressure may increase
Without further reduction in read-through events of alternative Poly+(A) vectors, this type of vector may prove unusable for selection and generation of high expressing cell lines

Method used

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  • Vectors and methods for recombinant protein expression
  • Vectors and methods for recombinant protein expression
  • Vectors and methods for recombinant protein expression

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Embodiment 1

[0041] In the present invention, the focus is on the effect of read-through events on the expression of selectable marker genes. It is believed that in a bicistronic configuration, read-through events with a frequency of 0.1-10% generate 0.1-1% bicistronic transcribed RNA in the total mRNA. McGrew (US Patent 6,632,637) reported an expression vector in which an internal polyadenylation signal is inserted between the DNA encoding the protein of interest and the DNA encoding the selectable marker, which allows a single promoter to produce a single cis Both antitronic messenger RNA (normal product) and dicistronic messenger RNA (readthrough product). Cells transfected with the alternative polyadenylation vector had approximately 8-fold higher IL-4R-specific messenger RNA (the protein of interest) than the control, and the amount of DHFR was 3.5-fold lower relative to the control, indicating that the frequency of 25% of the read-through events and produced 25% of the bicistronic m...

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Abstract

The present invention discloses a series of eukaryotic expression vectors utilizing the reduction of transcription read-through events to create stable and high-yield cell lines for recombinant protein expression. The vectors comprise more than one polyadenylation signal or one or more polyadenylation signals plus other DNA fragment which is known to enhance transcription termination to control the expression level of selection marker, with the configuration to transcribe the minimal level of full-length bicistronic mRNA to express the selection marker, which can be used to create stable cell lines at high expression levels, without the need for drug selection or drug mediated gene amplification.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to US Serial No. 61 / 367,661, filed July 26, 2010. The entire content and disclosure of the aforementioned applications are incorporated by reference into this application. technical field [0003] The present invention relates to the expression of recombinant proteins in eukaryotic cells. Background technique [0004] Expression of recombinant DNA in mammalian cells allows the production of a variety of complex glycosylated proteins (eg monoclonal antibodies) for clinical applications. Significant efforts are often required to establish cell lines that stably express such proteins at high levels (over 20 pg protein / cell / day). However, expression levels in most stable cell lines tend to be low (less than 1 pg / day / cell) and unstable (decrease over time and in large-scale cultures). Therefore, many individual clones need to be screened (often on the order of thousands of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC07K16/00C12N2510/00C12N2840/203C07K2317/14C12N2830/20C12N15/85
Inventor 吴晓云
Owner ADV BIOLOGICS
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