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Improved recombinant protein expression using a hybrid CHEF1 promoter

A CHEF1, promoter technology, used in the field of improved recombinant protein expression using hybrid CHEF1 promoters

Active Publication Date: 2015-12-30
CMC ICOS BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 recombinant protein expression using a hybrid CHEF1 promoter
  • Improved recombinant protein expression using a hybrid CHEF1 promoter
  • Improved recombinant protein expression using a hybrid CHEF1 promoter

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Embodiment approach

[0045] What is claimed is: 1. An expression vector comprising a Chinese hamster elongation factor-1α (CHEF1) transcriptional regulatory DNA and a cytomegalovirus (CMV) promoter and / or an adenovirus triplet leader (AdTPL) sequence.

[0046] 2. The expression vector of 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 that is 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 DNA between position 1 and position 11,716 in sequence ID NO: 1, or with position 1 and position 1 in sequence ID NO: 1. The DNA between positions 11,716 is at least 95% identical to a polynucleotide.

[0049] 5. The expression vector of embodiment 4, wherein the 5'CHEF1 transcriptional reg...

Embodiment

[0080] Gene sequences and expression vectors - 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 (Running Deer 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 a derived vector expressing Fc-glycoprotein fusion (GP1) and IgG1 antibody (MAb1) (Maniatis et al., J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. 545, 1982) and referred to the vector as pDEF38 -GP1, pDEF85-GP1, pDEF86-GP1, pDEF38-MAb1, pDEF85-MAb1 and pDEF86-MAb1.

[0081] Cell Line Construction - pDEF38-GP1, pDEF85-GP1, pDEF86-GP1, pDEF38-MAb1, pDEF85-MAb1, and pDEF86-MAb1 expression vectors were individually transfected into CHODG44 cells by standard electroporation methods, in the...

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Abstract

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

Description

[0001] This application contains the Sequence Listing in computer readable form (filename: 44744A_SeqListing.txt; size: 37,528 bytes; created: March 11, 2014) as a separate part of the disclosure, which is incorporated by reference in its entirety. into this article. [0002] CROSS-REFERENCE TO RELATED APPLICATIONS [0003] This application claims the benefit of priority from 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 recombinant protein expression through improvements in transcription, translation, protein folding and / or secretion is a fundamental priority for optimizin...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/67
CPCC07K14/4702C12N15/85
Inventor 霍华德·R·克拉可
Owner CMC ICOS BIOLOGICS INC