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
2001-01-12
2003-04-15
McKelvey, Terry (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...
C435S069700, C435S320100, C435S252000, C435S254100, C435S254200, C435S254300, C435S255100, C536S023100, C536S023400, C536S024100
Reexamination Certificate
active
06548274
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for producing polypeptides involving the use of a crippled translational initiator sequence operably linked to a gene encoding a selectable marker. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker.
2. Description of the Related Art
The continual development of new genetic engineering techniques has enabled the manipulation of the expression of genes encoding proteins. The manipulation of the coding region or the transcriptional control regions of a gene has frequently involved the isolation of the gene, manipulation of the nucleic acids contained in the gene in order to increase or decrease expression of the gene, and introduction of the manipulated gene into a suitable expression host.
A widely used method for increasing production of a polypeptide is to obtain a strain with multiple copies of the gene encoding the polypeptide through a process called amplification.
U.S. Pat. No. 5,578,461 discloses the inclusion via homologous recombination of an amplifiable selectable marker gene in tandem with a gene where strains containing amplified copies of the selectable marker in tandem with multiple copies of the gene can be selected for by culturing the strains in the presence of increasing amounts of the appropriate selectable agent.
WO 94/11523 discloses expression vectors comprising a fully impaired yeast Kozak consensus sequence for impairment of translation of a protein encoded by a dominant selectable marker.
It is an object of the present invention to provide improved methods for producing a polypeptide in a fungal host cell using crippled translational initiator sequences.
SUMMARY OF THE INVENTION
The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide; and (b) isolating the polypeptide from the cultivation medium; wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide in tandem with a second nucleic acid sequence comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker in which the 3′ end of the crippled translational initiator sequence is immediately upstream of the initiator codon of the gene encoding the selectable marker, wherein the crippled translational initiator sequence comprises a T at the −3 position and a T at one or more of the −1, −2, and −4 positions, and wherein the copy number of the first nucleic acid sequence has been increased by culturing the cell under conditions that select for multiple copies of the selectable marker.
The present invention also relates to methods for obtaining a fungal host cell for production of a polypeptide, comprising: (a) integrating into the genome of the fungal cell a nucleic acid construct comprising a first nucleic acid sequence encoding the polypeptide in tandem with a second nucleic acid sequence comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker in which the 3′ end of the crippled translational initiator sequence is immediately upstream of the initiator codon of the gene encoding the selectable marker; and (b) culturing the cell under conditions that select for multiple copies of the selectable marker wherein the copy number of the first nucleic acid sequence is increased, wherein the crippled translational initiator sequence comprises a T at the −3 position and a T at one or more of the −1, −2, and −4 positions.
The present invention also relates to nucleic acid constructs comprising a first nucleic acid sequence encoding the polypeptide in tandem with a second nucleic acid sequence comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker in which the 3′ end of the crippled translational initiator sequence is immediately upstream of the initiator codon of the gene encoding the selectable marker, wherein the crippled translational initiator sequence comprises a T at the −3 position and a T at one or more of the −1, −2, and −4 positions. The present invention further relates to recombinant expression vectors and fungal host cells containing such nucleic acid constructs.
REFERENCES:
patent: 5578461 (1996-11-01), Sherwin et al.
patent: 5648267 (1997-07-01), Reff
patent: 5821102 (1998-10-01), Berka et al.
patent: WO 94/11523 (1994-05-01), None
Bellini Daniel A.
Yaver Debbie S.
Leffers, Jr. Gerald G.
McKelvey Terry
Novozymes Biotech Inc.
Stames Robert L.
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