Artificial introns
The invention concerns the field of recombinant gene engineering. It concerns novel artificial introns and compositions comprising such introns as well as a method to improve expression of polypeptides from nucleic acids such as cloned genes, especially genes encoding antibodies and antibody derived fragments, and the production of various polypeptides in eukaryotic host cells using said novel artificial intron sequences.
1. A method of producing a heterologous protein of interest encoded by a polynucleotide sequence, wherein said method comprises the following steps:
a) introducing at least one artificial intron sequence into said polynucleotide sequence by
i. replacing a native intron sequence within said polynucleotide sequence, or
ii. by introducing at least one artificial intron sequence at the site of a native intron position,
b) introducing the polynucleotide sequence of step a) comprising the artificial intron sequence into a transcription unit or an expression vector,
c) transfecting a cell with said transcription unit or said vector of step b), and
d) cultivating said cell of step c) under conditions which allow expression of said protein of interest, wherein the protein of interest is an immunoglobulin a functional immunoglobulin fragment.
2. A method of producing a heterologous protein of interest encoded by a polynucleotide sequence, wherein said method comprises the following steps:
a) introducing at least one artificial intron sequence into a transcription unit or an expression vector comprising a polynucleotide sequence encoding the protein of interest by
i. replacing a native intron sequence within said polynucleotide sequence, or
ii. by introducing at least one artificial intron sequence at the site of a native intron position,
b) transfecting a cell with said transcription unit or expression vector of step a), and
c) cultivating said cell of step b) under conditions which avow expression of said protein of interest, and wherein the protein of interest is an immunoglobulin or a functional immunoglobulin fragment.
3. A method of producing a protein of interest encoded by a polynucleotide sequence, wherein said method comprises the following steps:
a) transfecting a cell with a transcription unit or expression vector comprising a gene of interest and comprising at least one artificial intron, whereby the artificial intron is
i. replacing a native intron sequence within said gene of interest, or
ii. positioned at the site of a native intron position within said gene of interest, wherein the gene of interest is an immunoglobulin gene, and
b) cultivating said cell of step a) under conditions which allow expression of said protein of interest.
4. The method according to claim 1 , wherein the artificial intron sequence is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:3, and wherein presence of the artificial intron sequence enhances expression of the gene of interest as compared to expression of gene of interest without the artificial intron sequence.
5. The method according to claim 4 , whereby the artificial intron sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
6. The method of claim 1 , additionally comprising the following step of isolating and purifying said protein of interest.
7. A method of generating a recombinant host cell or production cell comprising:
a) transfecting a cell with a transcription unit or expression vector comprising a gene of interest and comprising at least one artificial intron, whereby the artificial intron is
i. replacing a native intron sequence within said gene of interest, or
ii. positioned at the site of a native intron position within said gene of interest, wherein the gene of interest is an immunoglobulin gene, and
b) selecting a recombinant host cell or production cell that expresses the gene of interest.