Sandoz Ag

Novartis Ag

Boehringer Ingelheim Rcv Gmbh & Co. Kg

Clemens Achmüller

Johann Holzmann

Bernhard Auer

Philipp Wechner

Alois A Jungbauer

Rainer Hahn

Franz Hartner

Michael Fuchs

Waltraud Kaar

Ferdinand Zepeck

Hansjörg Toll

Michael Seifert

Stefan Hutwimmer

Florian Werther

Silvio Podmirseg

Thomas Specht

Matjaz Vogelsang

Norbert Lamping

Alois Jungbauer

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.

Multi-copy gene protein expression system

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.

Constitutive yeast LLP promotor-based expression systems

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.

DNA coding sequence for human G-CSF

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.

Sequence variants

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 XXXX, wherein Xto Xare amino acid residues and at least two of Xto Xis W, Y or F; b) peptides comprising the formula XXXX, wherein Xto Xare amino acid residues, at least one of Xto Xis W, and at least one of Xto Xis 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.

Affinity ligands

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 Ncomprising the replacement(s) by glutamic acid of one or more cysteines at positions corresponding to the positions 112, 134, and 138 of the autoprotease Nof classical swine fever virus and (b) the carboxyl-proximal fusion partner is an heterologous polypeptide of interest fused to the autoprotease Nso that it is capable of being cleaved from the fusion polypeptide by autoprotease Nautoproteolytic 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 isolated inclusion bodies, (iv) inducing autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide, and (v) isolating the cleaved heterologous polypeptide of interest.

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Kundl, Austria

Clemens Achmüller