Modification of cryptic splice sites in heterologous genes expressed in fungi
First Claim
1. A method for obtaining a recombinant fungal host cell, which produces a biologically active heterologous polypeptide comprising introducing into a fungal host cell a nucleic acid sequence encoding said heterologous polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%, wherein a recombinant fugal host cell which produces a biologically active heterologous polypeptide is obtained.
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Abstract
The present invention relates to methods for obtaining a fungal host cell comprising a nucleic acid sequence encoding a heterologous polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence. The present invention also relates to a nucleic acid sequence(s) with a modified cryptic splice site(s) as well as nucleic acid constructs, vectors, and host cells comprising said nucleic acid sequence(s). The present invention further relates to methods for recombinant production of a polypeptide encoded by said nucleic acid sequence.
12 Citations
25 Claims
- 1. A method for obtaining a recombinant fungal host cell, which produces a biologically active heterologous polypeptide comprising introducing into a fungal host cell a nucleic acid sequence encoding said heterologous polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%, wherein a recombinant fugal host cell which produces a biologically active heterologous polypeptide is obtained.
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16. A method for producing a biologically active polypeptide comprising
(a) introducing into a fungal host cell a nucleic acid sequence encoding said biologically active polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about70%; -
(b) cultivating the fungal host cell of step (a) in a nutrient medium; and (c) recovering said biologically active polypeptide from the nutrient medium of step (b). - View Dependent Claims (17)
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- 18. An isolated nucleic acid sequence with at least one cryptic splice site modified by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence and/or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%, said nucleic acid sequence encoding a biologically active polypeptide.
Specification