Combination of TIS sequence and signal peptide sequence for expressing a recombinant protein
The present invention relates to DNA constructs comprising a novel TIS sequence. The TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript. The DNA construct may further comprise a nucleic acid sequence encoding signal peptide. Additionally, the DNA constructs may also comprise a nucleic acid sequence encoding a recombinant protein or one or more polypeptide chains thereof. The invention further relates to an expression vector, expression cassette and host cell which comprise said DNA constructs. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.
1 . A DNA construct for expressing a recombinant protein in mammalian cells, wherein the DNA construct comprises:
a nucleic acid sequence of SEQ ID No. 1, wherein the nucleic acid sequence of SEQ ID No. 1 is a TIS sequence; and
a nucleic acid sequence which encodes a signal peptide, wherein the nucleic acid sequence which encodes the signal peptide comprises a nucleic acid sequence of SEQ ID No 2; and wherein the nucleic acid sequence of SEQ ID No. 1 comprises:
the ATG start codon in the nucleic acid sequence which encodes the first amino acid residue of the signal peptide; and
the first two nucleotides downstream of the ATG start codon in the nucleic acid sequence which encodes the second amino acid residue of the signal peptide.
2 . The DNA construct according to claim 1 , wherein the TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript.
3 . The DNA construct according to claim 1 , wherein the TIS sequence is a Kozak sequence which transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript.
4 . The DNA construct according to claim 1 , wherein the nucleic acid sequence of SEQ ID. No. 1 comprises:
6 nucleotides upstream of an ATG start codon; and
2 nucleotides downstream of an ATG start codon.
5 . The DNA construct according to claim 1 , wherein the nucleic acid sequence of SEQ ID No. 1 comprises:
the ATG start codon in SEQ ID No. 2; and
the first two nucleotides downstream of the ATG start codon in the nucleic acid sequence of SEQ ID No. 2.
6 . The DNA construct according to claim 1 , wherein the DNA construct comprises a nucleic acid sequence which encodes said recombinant protein, wherein said recombinant protein is an antibody, wherein said antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a fragment of said antibody.
7 . The DNA construct according to claim 6 , wherein the DNA construct which comprises the nucleic acid sequence which encodes said recombinant protein is an IgG4 monoclonal antibody.
8 . The DNA construct according to claim 1 , wherein the nucleic acid sequence which encodes a signal peptide is operably linked to the nucleic acid sequence which encodes the recombinant protein.
9 . The DNA construct according to claim 1 , wherein the nucleic acid sequence which encodes said recombinant protein comprises:
a first nucleic acid sequence which encodes a heavy chain of an antibody; and
a second nucleic acid sequence which encodes a light chain of an antibody.
10 . The DNA construct according to claim 9 , wherein the nucleic acid sequence which encodes a signal peptide is operably linked to:
a. the first nucleic acid sequence which encodes the heavy chain of an antibody; and/or
b. the second nucleic acid sequence which encodes the light chain of an antibody.
11 . An expression vector which comprises the DNA construct according to claim 1 .
12 . An expression cassette comprising the DNA construct according to claim 1 .
13 . A host cell which comprises a DNA construct according to claim 1 , wherein said host cell is a eukaryotic cell.
14 . The host cell according to claim 13 , wherein said host cell is a mammalian cell.
15 . A recombinant protein expressed by a DNA construct according to claim 1 , wherein said recombinant protein is an antibody or an antibody fragment thereof, a monoclonal antibody or a fragment thereof.
16 . An RNA expressed by the DNA construct according to claim 1 .
17 . A method of expressing a recombinant protein, comprising the steps of:
a. cloning one or more open reading frames encoding a recombinant protein, or one or more polypeptide chains thereof, into one or more DNA constructs according to claim 1 ; and
b. transfecting the resulting nucleic acid sequences into a host cell, wherein the host cell is a eukaryotic cell.
18 . The method according to claim 17 , further comprising the step of integrating the transfected nucleic acid sequence into the genome of the host cell.
19 . A DNA construct for expressing a signal peptide, wherein the DNA construct comprises a nucleic acid sequence which encodes a signal peptide, wherein the nucleic acid sequence which encodes a signal peptide comprises a nucleic acid sequence of SEQ ID No. 2.
20 . An expression vector which comprises the DNA construct according to claim 19 .
21 . An expression cassette comprising the DNA construct according to claim 19 .
22 . A host cell which comprises a DNA construct according to claim 19 .
23 . An RNA expressed by the DNA construct according to claim 19 .
24 . The recombinant protein according to claim 15 , wherein said monoclonal antibody is an IgG4 monoclonal antibody or fragment thereof.
25 . The method according to claim 17 , wherein said eukaryotic cell is a mammalian cell.