Heterologous intron within an immunoglobulin domain

1. A transcription unit or expression vector comprising a gene of interest encoding at least one domain of an immunoglobulin gene, comprising at least one heterologous intron located within an immunoglobulin exon, whereby the 5′ and 3′ ends of said exon are defined as occuring in a corresponding native immunoglobulin gene.

2. The transcription unit or expression vector of claim 1 , wherein said domain is a variable domain.

3. The transcription unit or expression vector according to claim 1 , wherein the domain of an immunoglobulin gene is the variable domain of the heavy chain (VH) or the variable domain of the light chain (VL).

4. The transcription unit or expression vector according to claim 1 , wherein said at least one heterologous intron is located within the framework 4 region of the variable immunoglobulin domain or between a variable and constant immunoglobulin domain.

5. The transcription unit or expression vector according to claim 1 , wherein said at least one heterologous intron is located within the framework 4 region of the VH domain at amino acid position 109, 111 or 113 according to Kabat numbering.

6. The transcription unit or expression vector according to claim 1 , wherein said at least one heterologous intron is located between the VL and the CL domain between amino acid positions 107 and 108 or within the framework 4 region between amino acid positions 103 and 104 according to Kabat numbering for the kappa chains or between amino acid positions 103 and 104 or at amino acid position 106 within the framework 4 region of the lambda chains according to Kabat numbering for the lambda chain.

7. The transcription unit or expression vector of claim 1 , wherein said domain is a constant domain of an immunoglobulin gene or a hinge region of an immunoglobulin gene.

8. The transcription unit or expression vector of claim 1 , wherein said gene/sequence encodes an Fc-fusion protein, a single chain format or another antibody-derived molecule.

9. The transcription unit or expression vector of claim 1 , wherein the heterologous intron is located at a position resulting in a functional splice donor site and a functional splice acceptor site.

10. The transcription unit or expression vector of claim 1 , wherein the heterologous intron is positioned within the nucleotide sequence successions CAG:C, CAG:T, AAG:C, AAG:T, TAG:T or TAG:C, preferably CAG:C, CAG:T, AAG:C or AAG:T, whereby the colons denote the site of intron insertion.

11. The transcription unit or expression vector of claim 10 , wherein the nucleotide sequence successions encode the amino acid pairs selected from the group consisting of:

CAG:C

CAG:T

AAG:C

AAG:T

TAG:C

TAG:T

GlnLeu

GlnPhe

LysLeu

LysPhe

PheSer

PheSer

CAGCTN

CAGTTY

AAGCTN

AAGTTY

TTTAGC

TTTAGT

GlnPro

GlnLeu

LysPro

LysLeu

SerSer

SerSer

CAGCCN

CAGTTR

AAGCCN

AAGTTR

TCTAGC

TCTAGT

GlnHis

GlnSer

LysHis

LysSer

TyrSer

TyrSer

CAGCAY

CAGTCN

AAGCAY

AAGTCN

TATAGC

TATAGT

GlnGln

GlnTyr

LysGln

LysTyr

CysSer

CysSer

CAGCAR

CAGTAY

AAGCAR

AAGTAY

TGTAGC

TGTAGT

GlnArg

GlnCys

LysArg

LysCys

LeuSer

LeuSer

CAGCGN

CAGTGY

AAGCGN

AAGTGY

CTTAGC

CTTAGT

SerAla

GlnTrp

GlnAla

LysTrp

ProSer

ProSer

TCAGCN

CAGTGG

CAAGCN

AAGTGG

CCTAGC

CCTAGT

ProAla

SerVal

LysAla

GlnVal

HisSer

HisSer

CCAGCN

TCAGTN

AAAGCN

CAAGTN

CATAGC

CATAGT

ThrAla

ProVal

GluAla

LysVal

ArgSer

ArgSer

ACAGCN

CCAGTN

GAAGCN

AAAGTN

CGTAGC

CGTAGT

AlaAla

ThrVal

LeuSer

GluVal

IleSer

IleSer

GCAGCN

ACAGTN

YTAAGC

GAAGTN

ATTAGC

ATTAGT

PheSer

AlaVal

SerSer

LeuSer

ThrSer

ThrSer

TTCAGC

GCAGTN

TCAAGC

YTAAGT

ACTAGC

ACTAGT

SerSer

PheSer

ProSer

SerSer

AsnSer

AsnSer

TCCAGC

TTCAGT

CCAAGC

TCAAGT

AATAGC

AATAGT

TyrSer

SerSer

GlnSer

ProSer

SerSer

SerSer

TACAGC

TCCAGT

CAAAGC

CCAAGT

AGTAGC

AGTAGT

CysSer

TyrSer

ArgSer

GlnSer

ValSer

ValSer

TGCAGC

TACAGT

MGAAGC

CAAAGT

GTTAGC

GTTAGT

LeuSer

CysSer

IleSer

ArgSer

AlaSer

AlaSer

CTCAGC

TGCAGT

ATAAGC

MGAAGT

GCTAGC

GCTAGT

ProSer

LeuSer

ThrSer

IleSer

AspSer

AspSer

CCCAGC

CTCAGT

ACAAGC

ATAAGT

GATAGC

GATAGT

HisSer

ProSer

LysSer

ThrSer

GlySer

GlySer

CACAGC

CCCAGT

AAAAGC

ACAAGT

GGTAGC

GGTAGT

ArgSer

HisSer

ValSer

LysSer

LeuAla

LeuVal

CGCAGC

CACAGT

GTAAGC

AAAAGT

YTAGCN

YTAGTN

IleSer

ArgSer

AlaSer

ValSer

IleAla

IleVal

ATCAGC

CGCAGT

GCAAGC

GTAAGT

ATAGCN

ATAGTN

ThrSer

IleSer

GluSer

AlaSer

ValAla

ValVal

ACCAGC

ATCAGT

GAAAGC

GCAAGT

GTAGCN

GTAGTN

AsnSer

ThrSer

GlySer

GluSer

AACAGC

ACCAGT

GGAAGC

GAAAGT

SerSer

AsnSer

GlySer

AGCAGC

AACAGT

GGAAGT

ValSer

SerSer

GTCAGC

AGCAGT

AlaSer

ValSer

GCCAGC

GTCAGT

AspSer

AlaSer

GACAGC

GCCAGT

GlySer

AspSer

GGCAGC

GACAGT

GlySer

GGCAGT.

colon: site of intron insertion, N = any base, Y = C or T, R = A or G, M = C or A

12. The transcription unit or expression vector of claim 1 , wherein the heterologous intron is a nucleic acid sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

13. The transcription unit or expression vector according to claim 1 , wherein said transcription unit or expression vector further comprises at least one promoter and at least one terminator, optionally said transcription unit or expression vector further comprises at least one selection marker and/or at least one enhancer.

14. A host cell comprising the transcription unit or expression vector according to claim 1 .

15. The host cell of claim 14 , wherein said cell is a eukaryotic cell.

16. A method of producing a protein of interest encoded by a polynucleotide sequence comprising at least one domain of an immunoglobulin gene comprising:

a) introducing at least one heterologous intron sequence into said domain by placing the intron into a nucleotide sequence of an immunoglobulin exon, whereby the 5′ and 3′ ends of the exon are defined as occuring in a corresponding native immunoglobulin gene, and

b) introducing the immunoglobulin domain comprising/containing the nucleic acid sequence of step a) into a transciption unit or a expression vector, and

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.

17. A method of producing a protein of interest encoded by a polynucleotide sequence comprising at least one domain of an immunoglobulin gene comprising:

a) transfecting a cell with the transcription unit or expression vector of claim 1 ,

b) cultivating said cell of step a) under conditions which allow expression of said protein of interest.

18. The method according to claim 16 , whereby the heterologous intron sequence is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

19. The method of claim 16 , additionally comprising the following step of isolating and purifying said protein of interest.

20. A method of generating a recombinant host cell/production cell comprising:

a) transfecting a cell with the transcription unit or expression vector of claim 1 , and

b) selecting a recombinant host cell/production cell.

21. A kit comprising

a vector including at least one intron sequence flanked by suitable recognition sites for restriction enzymes allowing the integration of a first and/or a second nucleotide sequence coding for at least a part of an immunoglobulin gene, and

instructions and optionally a vector map, and

optionally a host cell, and

optionally a cultivation medium for the cultivation of a host cell, and/or

optionally a selection medium for selecting and cultivating a transfected host cell.

22. A kit comprising

a) a vector including at least parts of a transcription unit comprising a first nucleotide sequence comprising at least one heterologous intron located within an immunoglobulin exon or parts of an immunoglobulin exon, whereby the 5′ and 3′ ends of said exon are defined as occuring in a corresponding native immunoglobulin gene sequence, and whereby the nucleotide sequence of this transciption unit comprises suitable recognition sites for restriction enzymes allowing the integration of a second nucleotide sequence coding for another part of the gene of interest, whereby the two nucleotide sequences are operatively linked, and

b) instructions and optionally a vector map, and

c) optionally a host cell, and

d) optionally a cultivation medium for the cultivation of a host cell, and/or

e) optionally a selection medium for selecting and cultivating a transfected host cell.