Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-06-25
2001-11-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254300, C435S320100, C435S471000, C435S243000, C435S455000, C435S468000, C536S023100
Reexamination Certificate
active
06323002
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for modifying the production of a polypeptide by a cell.
2. Description of the Related Art
Several methods have been used to modify the production of polypeptides by mutagenizing cells. For example, the production of proteins has been altered by producing mutant cells by classical mutagenesis which involves treating cells with chemical, physical, and biological agents as mutagenic (mutation inducing) agents to increase the frequency of mutational events.
Production of proteins also has been modified by mutagenesis of a cell with short sections of double-stranded DNA, consisting of more than 2000 base pairs, called transposons which usually code for resistance to one or sometimes several antibiotics. Transposons are able to move or jump within the genome, even between a bacterial chromosome and a plasmid, and they are able to become integrated in a number of different sites on the genome. An insertion of a transposon within a structural gene interrupts the normal nucleotide sequence of the gene so that it can no longer deliver the information for the synthesis of the normal, functional polypeptide (Seifert et al., 1986
, Proceedings of the National Academy of Sciences USA
83: 735-739). An insertion also may disrupt a gene whose gene product is required for expression (Márquez-Magaña and Chamberlin, 1994
, Journal of Bacteriology
176: 2427-2434). In addition, Errede et al. (1980
, Cell
22: 427-436) disclose the insertion of a transposable element adjacent to the structural gene coding for iso-2-cytochrome c causing overproduction. Furthermore, WO 96/29414 discloses that transposable elements may be constructed containing a transposon and a DNA sequence capable of regulating a targeted gene where upon introduction into a cell the transposable element integrates into the genome of the cell in a manner which regulates the expression of the gene.
A widely used method for increasing production of a polypeptide is amplification to produce multiple copies of the gene encoding the polypeptide. For example, U.S. Pat. No. 5,578,461 discloses the inclusion via homologous recombination of an amplifiable selectable marker gene in tandem with the gene where cells containing amplified copies of the selectable marker can be selected for by culturing the cells in the presence of the appropriate selectable agent.
In addition, the production of polypeptides has been increased by replacing one promoter with a different promoter or one signal peptide coding region with another. See, e.g., U.S. Pat. No. 5,641,670.
Methods for altering gene expression by disrupting genes encoding various regulatory elements have also been described. For example, Toma et al. (1986
, Journal of Bacteriology
167: 740-743) showed that a deletion from −156 to −90 in the npr promoter region caused overexpression of the neutral protease encoded by the npr gene. Pero and Sloma (1993, In A. L. Sonensheim, J. A. Hoch, and R. Losick, editors,
Bacillus subtilis
and
Other Gram-Positive Bacteria
, pp. 939-952, American Society for Microbiology, Washington, D.C.) disclose that mutating the sporulation gene spoOA results in deficient synthesis of proteases and that mutations in the abrB gene restore synthesis.
The production of polypeptides also has been increased by disrupting DNA sequences encoding a protease capable of hydrolyzing the polypeptide under the conditions for producing the polypeptide.
The secretion of polypeptides has also been modified by overproduction of secretion proteins (Ruohonen et al., 1997
, Yeast
, 3: 337-351), and producing a super-secreting cell (U.S. Pat. No. 5.312,735).
Methods for increasing the production of metabolites have also been described. For example, WO 96/41886 discloses that increased production of clavam produced by an organism having at least part of the clavam pathway and at least part of a cephalosporin pathway by interfering with the conversion of L-lysine to L-alpha-aminoadipic acid in the cephalosporin pathway. WO 94/13813 discloses the disruption of gene which encodes a protein which degrades betaine, an enzyme inducer.
It is an object of the present invention to provide new and improved methodologies for altering production of polypeptides and metabolites.
BRIEF SUMMARY OF THE INVENTION
The present invention is drawn to methods for modifying the production of a polypeptide by a cell. In the methods of the present invention, a nucleic acid construct is introduced into a cell which contains a DNA sequence encoding a specific polypeptide. The introduced nucleic acid construct integrates into the host genome at a locus not within the DNA sequence of interest to produce a mutant cell. The integration of the nucleic acid construct into the locus modifies the production of the polypeptide by the mutant cell relative to the parent cell. Mutant cells are then identified in which the polypeptide's production is modified by the mutant cell relative to the parent cell. Modification is determined by comparing production of the polypeptide when the mutant cell and the parent cell are cultured under the same conditions.
An advantage of the present invention is that the mutation can be recovered and leads to a modification of the production of a polypeptide encoded by a DNA sequence which does not contain the mutation.
REFERENCES:
Studier, et al. Journal of Molecular Biology, pp37-44, 1991.*
Robyt, et al. “Biochemical Techniques: Theory and Practice” chapters 4,5, and 8-10, 1991.
Brody Howard
Hansen Kim
Lamsa Michael
Yaver Deborah S.
Guzo David
Leffeis, Jr. Gerald G.
Novozymes Biotech, Inc
Starnes Robert L.
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