Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-02-25
2000-10-10
Brusca, John S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 9142, 435455, 4353201, 536 235, C12N 1567, C12N 1579, C12N 510, C12N 1512
Patent
active
061300624
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates generally to improved production of proteins in host cells. In particular it relates to improved production of proteins which are required for therapeutic or reagent applications and which are difficult to obtain in sufficient quantities. The invention also relates to a method of producing recombinant nucleic acid molecules which encode said proteins, to the recombinant nucleic acid molecules and recombinant constructs comprising the same and to the proteins produced thereby. Of particular interest are nucleic acid molecules encoding hormones, cytokines, growth factors, receptors and their ligands and cell surface proteins including complement regulating proteins.
BACKGROUND OF THE INVENTION
Although many genes and cDNAs encoding useful proteins have been isolated, a major problem facing scientists is the production of sufficient amounts of recombinant proteins for further study and for diagnostic or therapeutic applications. This is particularly so where commercial production of the protein is desired. In particular the production of hormones, cytokines, growth factors, receptors and their ligands and other proteins by recombinant means has become a major endeavour.
By way of example the cell surface molecule, CD46 (also known as membrane cofactor or MCP) has not been expressed in eukaryotic hosts as well as other cDNA constructs using a variety of expression vectors. Similar concerns have arisen about CD55 (DAF) and CD35 (CR1) and factor H which like CD46, are members of the family of proteins called Regulators of Complement Activation (RCA).
Regulators of Complement Activation control the amplification of the complement cascade. Each of the members of this family share structural characteristics, principally the .about.60-65 amino acid Short Consensus Repeat (SCR) modules, which are responsible for complement binding and regulatory functions. Genes encoding the family members are linked and map to chromosome 1q3.2.
The biology of these molecules is of interest in inflammation and homeostasis. However, there is currently a further application for them in the potential use of porcine organs or other organs for grafting to humans. Such xenogenic organs undergo antibody and complement mediated hyperacute rejection.
A limitation to studies of CD46 and other RCAs has been that on transfection with constructs encoding the recombinant proteins in either transient or stable systems very few cells have been transfected and those that are express low amounts of protein. Indeed, in the inventors' laboratory and others, sophisticated cell sorting, cloning and selection procedures have been necessary to isolate appropriate cells. This, in particular, has created problems with the analysis of recombinant CD46, its physiological role and value as a therapeutic agent.
In work leading up to the present invention the inventors have surprisingly found that alterations in a nucleic acid encoding CD46 led to high levels of CD46 production in a transfected eukarotic host. Protein production levels of up to 8 times that obtained from the wildtype gene for CD46 have been observed. These alterations comprised reducing the amount or proportion of adenine (A) and/or thymine (T) in an A and/or T rich region in the nucleic acid.
SUMMARY OF THE INVENTION
The present invention provides a method of increasing or improving mammalian protein production, without demonstrably altering RNA stability, in a host cell where a nucleic acid encoding the mammalian protein has an A and/or T rich region present in an exon, said method comprising altering the nucleic acid by reducing or lowering the amount of A and/or T in said region and transfecting a host cell with said nucleic acid and, under appropriate conditions, obtaining expression of said nucleic acid.
In another aspect the present invention provides a method for enhancing production of RCA proteins in a host cell where a nucleic acid encoding an RCA has an A and/or T rich region present in one or more of its exons said method com
REFERENCES:
Lusky et al. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature vol. 293 pp. 79-81, 1981.
Christiansen Dale
Loveland Bruce
McKenzie Ian F. C.
Milland Julie
Brusca John S.
The Austin Research Institute
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