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Improved expression of recombinant proteins using a hybrid Chef1 promoter

A CHEF1, promoter technology, applied in the field of improving recombinant protein expression using a hybrid CHEF1 promoter

Active Publication Date: 2019-03-29
CMC ICOS BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the CHEF1 vector has proven successful in driving high-level expression of recombinant proteins, there is a continuing need to develop improved expression systems

Method used

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  • Improved expression of recombinant proteins using a hybrid Chef1 promoter
  • Improved expression of recombinant proteins using a hybrid Chef1 promoter
  • Improved expression of recombinant proteins using a hybrid Chef1 promoter

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Embodiment approach

[0045] 1. An expression vector comprising Chinese hamster elongation factor-1α (CHEF1) transcriptional regulatory DNA and cytomegalovirus (CMV) promoter and / or adenovirus tripartite leader (AdTPL) sequence.

[0046] 2. The expression vector according to embodiment 1, wherein the CHEF1 transcriptional regulatory DNA comprises a 5'CHEF transcriptional regulatory DNA.

[0047] 3. The expression vector of embodiment 2, wherein the 5'CHEF1 transcriptional regulatory DNA comprises Sequence ID NO:1 or a polynucleotide at least 95% identical to Sequence ID NO:1.

[0048] 4. The expression vector of embodiment 2, wherein the 5'CHEF1 transcriptional regulatory DNA comprises a DNA between position 1 and position 11,716 in sequence ID NO:1, or a DNA located between position 1 in sequence ID NO:1 A polynucleotide that is at least 95% identical to the DNA between positions 11,716.

[0049] 5. The expression vector according to embodiment 4, wherein the 5'CHEF1 transcriptional regulatory DN...

Embodiment

[0080] Gene sequences and expression vectors—the DNA fragments encoding the CMV promoter (SEQ ID NO:4) and the CMV-AdTPL promoter (SEQ ID NO:5) were chemically synthesized and cloned into the aforementioned CHEF1 expression vector pDEF38 (RunningDeer and Allison , 2004), thereby creating a CHEF1-CMV-promoter vector called pDEF85 ( figure 1 ), and the CHEF1-CMV-AdTPL promoter vector called pDEF86 ( figure 2 ). Standard molecular biology techniques were used to create derivative vectors expressing Fc-glycoprotein fusions (GP1) and IgG1 antibodies (MAb1) (Maniatis et al., J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. 545, 1982) and the resulting The vectors are referred to as pDEF38-GP1, pDEF85-GP1, pDEF86-GP1, pDEF38-MAb1, pDEF85-MAb1 and pDEF86-MAb1.

[0081] Cell line construction—transfect pDEF38-GP1, pDEF85-GP1, pDEF86-GP1, pDEF38-MAb1, pDEF85-MAb1, and pDEF86-MAb1 expression vectors into CHO DG44 cells individually by standard electroporation ...

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Abstract

The present invention provides expression vectors and host cells for high-level expression of recombinant proteins. The expression vector comprises a Chinese hamster ovary elongation factor 1-alpha (CHEF1) transcriptional regulatory DNA element, a cytomegalovirus (CMV) promoter and / or a human adenovirus tripartite leader (AdTPL) sequence. The present invention achieves increased protein expression and better yields of host cells compared to previously described expression systems.

Description

[0001] This application contains as a separate part of the disclosure a Sequence Listing in computer readable form (Filename: 44744A_SeqListing.txt; Size: 37,528 bytes; Created: March 11, 2014), which is incorporated by reference in its entirety and into this article. [0002] Cross References to Related Applications [0003] This application claims the benefit of priority to US Provisional Patent Application Serial No. 61 / 777,603, filed March 12, 2013. The disclosure of said priority application is incorporated herein by reference. technical field [0004] The present invention is directed to expression vectors comprising novel promoter-enhancer combinations that enhance heterologous protein expression and have practical application in the field of recombinant protein production. Background technique [0005] Increased expression of recombinant proteins through improvements in transcription, translation, protein folding and / or secretion is a substantial priority for optim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/67
CPCC07K14/4702C12N15/85
Inventor 霍华德·R·克拉可
Owner CMC ICOS BIOLOGICS INC