Methods and compositions for high protein production from non-native DNA
AssigneeCreative BioMolecules, Inc.
InventorsHaimanti Dorai, Herrmann Oppermann
Life of this patent
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Abstract
Disclosed herein are improved methods and compositions for achieving enhanced protein production expressed from non-native gene constructs, including single chain sFv and derivative sequences. The methods and compositions are particularly useful for creating stably transfected, contitutively expressing immortalized mammalian cell lines that exhibit high recombinant protein productivity while maintaining a low copy number per cell of the non-native recombinant DNA sequence encoding the protein of interest.
Claims26
A method of manufacturing a cell line for the enhanced production of a biosynthetic single chain binding protein, the method comprising the steps of:
- (a) transfecting an immortalized eukaryotic cell with a nucleic acid comprising a DNA sequence encoding a viral transcription activator protein that acts on and stimulates a viral promoter, wherein said DNA sequence encoding said viral transcription activator protein is operatively associated with, and under the control of, a DNA sequence defining a transcription promoter sequence which can induce transcription of said viral transcription activator protein DNA sequence, said transcription promoter DNA sequence being selected to permit unlimited or limited transcription of said viral transcription activator protein DNA sequence;
- (b) transfecting said immortalized eukaryotic cell with nucleic acids comprising:
- (1) a DNA sequence encoding a viral transcription promoter operatively associated with a non-native reporter DNA sequence encoding a biosynthetic single chain binding protein, said viral transcription promoter capable of being stimulated by said viral transcription activator protein, and competent to act on and induce transcription of said reporter DNA sequence to produce an RNA transcript, said transcript, when translated, producing a single-chain binding protein that, when properly folded, has a structural conformation capable of mono- or bifunctional binding activity;
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- (2) a DNA sequence encoding an RNA sequence, said RNA sequence promoting translation of said RNA transcript from step (b)(1) above;
- (c) culturing said transfected cell under conditions that allow said transfected cell to integrate into its genome said DNA sequences such that said integration permits growth of said cell and production of said single-chain binding protein, said protein being produced at a level greater than if said cell is transfected with said reporter DNA sequence alone or in combination with only one of the DNA sequences of step (a) or (b)(2) above;
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- (d) identifying a clone of said cultured transfected cell of step (c) that expresses at least 1 .mu.g single-chain binding proteins/10.sup.6 cell/ml.
The method of manufacture of claim 27 wherein said transcription promoter DNA sequence is selected to limit transcription of said viral transcription activator DNA sequence.
Dependencies are inferred from the claim text.