Abstract: The present invention belongs to the field of biotechnology, specifically to the field of recombinant protein expression. The present invention focuses on two problems commonly encountered during recombinant protein expression, low quantity of protein expression and genetic instability of cell lines used for recombinant protein expression. The basic principle of the present invention is to introduce several expression cassettes into a cell which expression cassettes all code for the same mature recombinant protein of interest, but which expression cassettes have different nucleotide sequences. Expression cassette means a polynucleotide sequence which comprises at least a promoter sequence, a start codon, a polynucleotide sequence coding for a protein which is intended to be recombinant expressed (POI), a stop codon and a terminator.

Abstract: The present invention belongs to the field of biotechnology, recombinant protein production, molecular biology, microbiology and microbial genetics. It provides a modified eukaryotic cell that is modified to the effect that the modified eukaryotic cell is not able to provide an SSN6-like related protein or an SSN6-like protein that exerts its wildtype function and/or wildtype activity, the amount of SSN6-like related protein or of SSN6-like protein being present in the modified eukaryotic cell differs from the amount of SSN6-like related protein or of SSN6-like protein being present in its wildtype form, and/or essentially no SSN6-like related protein or SSN6-like protein is present in the modified cell. Additionally, the present invention provides a polynucleotide sequence comprising a modified ssn6-like related gene or modified ssn6-like gene, and a vector comprising said polynucleoptide.

Abstract: The present invention belongs to the field of biotechnology, specifically to the field of recombinant protein expression. The present invention focuses on two problems commonly encountered during recombinant protein expression, low quantity of protein expression and genetic instability of cell lines used for recombinant protein expression. The basic principle of the present invention is to introduce several expression cassettes into a cell which expression cassettes all code for the same mature recombinant protein of interest, but which expression cassettes have different nucleotide sequences. Expression cassette means a polynucleotide sequence which comprises at least a promoter sequence, a start codon, a polynucleotide sequence coding for a protein which is intended to be recombinant expressed (POI), a stop codon and a terminator.

Abstract: The present invention provides a modified eukaryotic cell wherein the modified eukaryotic cell is not able to provide an SSN6-like protein that exerts its wildtype function and/or wildtype activity, the amount of SSN6-like protein being present in the modified eukaryotic cell differs from the amount of SSN6-like protein being present in its wildtype form, and/or essentially no SSN6-like protein is present in the modified cell. Additionally, the present invention provides a polynucleotide sequence comprising a modified ssn6-like gene, and a vector comprising said polynucleoptide. Additionally provided is an expression vector comprising a promoter that is repressed in the presence of SSN6-like protein, and a host cell comprising said vectors. The present invention further refers to a method for determining the purity of a composition by using the modified eukaryotic cell, to a method of expressing gene(s) of interest, and eukaryotic cells comprising modified ssn6-like gene.

Abstract: The present invention relates in general to a nucleic acid encoding human granulocyte-colony stimulating factor (G-CSF), wherein the first leucine residue occurring on the N-terminal end of the encoded G-CSF is encoded by a codon other than the CTG/CUG codon, and wherein the nucleic acid does neither comprise the nucleic acid sequence according to SEQ ID NO: 1, nor according to SEQ ID NO: 2, nor according to SEQ ID NO: 3, nor according to SEQ ID NO: 4. The present invention also relates to a nucleic acid 100% complementary to the aforementioned nucleic acid, as well as to vectors and host cells comprising the aforementioned nucleic acids. Finally, the present invention relates to methods for producing human G-CSF using these nucleic acids, vectors and/or host cells and resulting G-CSF compositions.

Abstract: Amino acid residue misincorporations are necessarily found in sequence variants at low concentrations in admixture with expressed polypeptides, resulting from one or more base mismatches within codons susceptible to amino acid residue misincorporation during transcription and/or translation. The invention provides a method of optimizing the coding sequences of a polynucleotide that encodes a polypeptide, wherein at least one codon is susceptible to amino acid residue misincorporation. The method of the invention can be used to reverse-engineer an unknown coding sequence, which encodes the same polypeptide, but differs in said at least one codon from the known coding sequence. The method can further be used to alter the immunogenic potential of an expressed polypeptide. Thus, the invention is useful in engineering optimized polynucleotides encoding polypeptides.

Abstract: The present invention relates in general to a nucleic acid encoding human granulocyte-colony stimulating factor (G-CSF), wherein the first leucine residue occurring on the N-terminal end of the encoded G-CSF is encoded by a codon other than the CTG/CUG codon, and wherein the nucleic acid does neither comprise the nucleic acid sequence according to SEQ ID NO: 1, nor according to SEQ ID NO: 2, nor according to SEQ ID NO: 3, nor according to SEQ ID NO: 4. The present invention also relates to a nucleic acid 100% complementary to the aforementioned nucleic acid, as well as to vectors and host cells comprising the aforementioned nucleic acids. Finally, the present invention relates to methods for producing human G-CSF using these nucleic acids, vectors and/or host cells and resulting G-CSF compositions.

Abstract: The present invention belongs to the field of biotechnology, recombinant protein production, molecular biology, microbiology and microbial genetics. It provides a modified eukaryotic cell that is modified to the effect that the modified eukaryotic cell is not able to provide an SSN6-like related protein or an SSN6-like protein that exerts its wildtype function and/or wildtype activity, the amount of SSN6-like related protein or of SSN6-like protein being present in the modified eukaryotic cell differs from the amount of SSN6-like related protein or of SSN6-like protein being present in its wildtype form, and/or essentially no SSN6-like related protein or SSN6-like protein is present in the modified cell. Additionally, the present invention provides a polynucleotide sequence comprising a modified ssn6-like related gene or modified ssn6-like gene, and a vector comprising said polynucleoptide.

Abstract: Amino acid residue misincorporations are necessarily found in sequence variants at low concentrations in admixture with expressed polypeptides, resulting from one or more base mismatches within codons susceptible to amino acid residue misincorporation during transcription and/or translation. The invention provides a method of optimizing the coding sequences of a polynucleotide that encodes a polypeptide, wherein at least one codon is susceptible to amino acid residue misincorporation. The method of the invention can be used to reverse-engineer an unknown coding sequence, which encodes the same polypeptide, but differs in said at least one codon from the known coding sequence. The method can further be used to alter the immunogenic potential of an expressed polypeptide. Thus, the invention is useful in engineering optimized polynucleotides encoding polypeptides.

Abstract: Disclosed is an affinity matrix comprising a solid phase and an affinity ligand comprising peptide bonds coupled to this solid phase, wherein the affinity ligand comprising peptide bond is selected from the following group of ligands: a) peptides comprising the formula X1X2X3X4, wherein X1 to X4 are amino acid residues and at least two of X1 to X4 is W, Y or F; b) peptides comprising the formula X5X6X7X8, wherein X5 to X8 are amino acid residues, at least one of X5 to X8 is W, and at least one of X5 to X8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.

Abstract: The invention relates to a process for the recombinant production of a heterologous polypeptide of interest by cultivating a bacterial host cell transformed with an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide wherein (a) the amino-proximal fusion partner is an autoprotease Npro comprising the replacement(s) by glutamic acid of one or more cysteines at positions corresponding to the positions 112, 134, and 138 of the autoprotease Npro of classical swine fever virus and (b) the carboxyl-proximal fusion partner is an heterologous polypeptide of interest fused to the autoprotease Npro so that it is capable of being cleaved from the fusion polypeptide by autoprotease Npro autoproteolytic activity, said process comprising (i) cultivating the transformed host cell under conditions permitting the expression of the fusion polypeptide and the formation of corresponding cytoplasmic inclusion bodies, (ii) isolating the inclusion bodies from the host cell, (iii) solubilizing the isol

Abstract: The invention relates to a process for the recombinant production of a heterologous polypeptide of interest, comprising, (i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion polypeptide, the fusion polypeptide comprising a derivative of an autoprotease Npro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease Npro of Pestivirus is replaced by another amino acid residue, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such, that the second polypeptide is capable of being cleaved from the fusion polypeptide by the autoproteolytic activity of the first polypeptide, said second polypeptide being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion polypeptide and formation of corresponding cytoplasmic inclusion bodies, (ii) isolation of the inclus

Abstract: Disclosed is an affinity matrix comprising a solid phase and an affinity ligand comprising peptide bonds coupled to this solid phase, wherein the affinity ligand comprising peptide bond is selected from the following group of ligands: a) peptides comprising the formula X1X2X3X4, wherein X1 to X4 are amino acid residues and at least two of X1 to X4 is W, Y or F; b) peptides comprising the formula X5X6X7X8, wherein X5 to X8 are amino acid residues, at least one of X5 to X8 is W, and at least one of X5 to X8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.