EP4146687A1 - Expression of collagen peptide components in procaryotic systems - Google Patents
Expression of collagen peptide components in procaryotic systemsInfo
- Publication number
- EP4146687A1 EP4146687A1 EP21724247.8A EP21724247A EP4146687A1 EP 4146687 A1 EP4146687 A1 EP 4146687A1 EP 21724247 A EP21724247 A EP 21724247A EP 4146687 A1 EP4146687 A1 EP 4146687A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- collagen peptide
- collagen
- nucleotide sequence
- proline
- peptide component
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 239000012588 trypsin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to processes for the production of hydroxylated collagen peptide components and the hydroxylated collagen peptide components obtained from these processes, in particular hydroxylated collagen peptides.
- Collagen is the most common protein in the human body and, as the main component of the extracellular matrix, gives various tissues their characteristic flexibility and elasticity. Collagen polypeptide chains form a helix structure, which is caused by the repetitive consensus sequence of the amino acids (Gly-XY) n , where "Gly” is for glycine and "X” and “Y” are each for any one Amino acid stand.
- the most common repetitive unit in collagen is Gly-Pro-Hyp, where “Pro” stands for the amino acid proline and “Hyp” is hydroxyproline ((S) - (-) - irans-4-hydroxyproline).
- the degree of hydroxylation of proline in human collagens varies between approx. 42-54%. In the case of bovine collagen, the degree of hydroxylation is in the region of 45%.
- Collagen is an important source of biologically active peptides with a wide range of applications. Due to their high antioxidant and antihypertensive activity in combination with their low antigenicity, collagen peptides are particularly suitable for use as "functional food". In terms of sustainability, it is desirable to recombinantly produce collagen peptides, especially from mammals such as cattle (Bos taurus).
- mammals such as cattle (Bos taurus).
- bovine origin, preferably peptides or fragments from the natural sequence of a collagen polypeptide chain, in particular ⁇ -1 type I collagen (CollAl)
- different cell-based expression systems are possible in principle : Prokaryotes such as Escherichia coli (E. coli) and eukaryotic organisms such as yeast, plants, mammalian cells and insect cells.
- bacteria and yeasts do not have natural mechanisms for hydroxylating proline, they enable the cost-efficient production of recombinant proteins.
- prokaryotic systems such as E. coli
- E. coli for the industrial production of recombinant collagen peptides
- these are characterized in many ways, are genetically easily accessible, have a low complexity, have a high specific growth rate, can be attracted to high cell densities (high cell density fermentation), grow on inexpensive culture media, enable rapid expression of recombinant proteins with a high level of expression and often see high space in prokaryotic systems -Time yields can be achieved.
- the bovine Coli Al (bCollAl) only has a sequence identity of 62.1% and a sequence similarity of 67.5% to the human Col3Al, which results in a different behavior with regard to the expression of bovine collagen Coli Al and that in the prior art described cytosolic expression approach to the recombinant expression of the bovine collagen Coli Al in prokaryotic systems cannot be transferred.
- the production of recombinant collagen peptides, in particular collagen peptides of bovine origin, with the help of prokaryotic systems, for example with the host organism Escherichia coli, is a particular challenge, since collagen peptides can also have antimicrobial effects in addition to the desired biological activity.
- Some naturally occurring collagen peptides such as bovine CollAl, have already successfully expressed collagens in E. coli in comparison to other prior art - such as marine collagens, collagen-like proteins of bacterial or artificial origin and so-called designers -Collagens, for example consisting of repetitive GEK (G: glycine, E: glutamic acid, K: lysine) and GDK sequences (D: aspartic acid) and non-naturally occurring amino acid sequences (no 100% match to natural collagen such as CollAl) - one higher hydrophobicity (33-43%) and / or a higher proline content (20-31%). Because of the antimicrobial effect brought about by this, the recombinant expression of many collagen peptides in prokaryotic systems therefore proves to be problematic.
- bovine P4H has only a low degree of homology to human P4H.
- the bovine ⁇ subunit has a sequence identity of approx. 37% and a sequence similarity of approx. 57% to the human a subunit and the bovine ⁇ subunit has a sequence identity of approx. 33% and a sequence similarity of approx 50% to the human ß-subunit.
- the problem with the recombinant expression of all P4Hs from vertebrates in prokaryotes is that only the a2ß2 tetramer with a comparatively very high molecular weight of about 240 kDa has catalytic activity and the formation of the native structure of the a subunit and the association with the tetramer intramolecular disulfide bridges is necessary.
- An in vitro association of the subunits to the tetramer does not work and the co-expression of the ß-subunit is necessary to keep the a-subunit in soluble form.
- Another problem is that the activity and stability of the tetramer depends heavily on the availability of collagen substrates, which makes the production process and the expression / induction strategy very difficult.
- the present invention is therefore based on the technical problem of providing a method for producing a recombinantly produced hydroxylated collagen peptide component, in particular recombinant hydroxylated collagen peptides, which overcomes the aforementioned disadvantages, which in particular allows recombinant hydroxylated collagen peptide components, in particular recombinant hydroxylated collagen peptides, too on a larger industrial and cost-effective scale, see systems in prokaryoti.
- the present invention is based, in particular, on the technical problem of providing a method for producing a recombinant hydroxylated collagen peptide component with a collagen-typical hydroxylation pattern in prokaryotic systems.
- the present invention solves the technical problem on which it is based by means of the subject matter of the independent claims, in particular by providing methods for producing a recombinant hydroxylated collagen peptide component according to the present invention.
- the present invention relates to a method for producing a recombinant collagen peptide component in prokaryotic see systems, comprising the steps: a) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence encoding at least one recombinant collagen peptide component, the at least one recombinant collagen peptide component coding nucleotide sequence comprises the nucleotide sequence of at least one collagen peptide, b) culturing the prokaryotic see expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component, the collagen peptide component compared to the at least one from the collagen peptide Component-encoding nucleotide sequence-encoded collagen peptide has a reduced hydrophobicity and / or a reduced prolin content, c) recovery of the collagen peptide component.
- the present invention is based in particular on the identification of the combination of the proline content and the hydrophobicity as decisive influencing factors on the antimicrobial effectiveness of some collagen peptides.
- the hydrophobic character of the collagen peptides presumably leads to the storage of the peptides in the bacterial membranes and thus to the loss of membrane integrity, as is the case with many extracellularly applied antimicrobial peptides.
- the high prolin content of the collagen peptides presumably leads to a disruption of cell metabolism at the level of protein synthesis without disrupting the cell membrane.
- the method according to the invention is based on a prokaryotic expression system, preferably E.
- the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, that is to say that the nucleotide sequence coding for at least one recombinant collagen peptide component consists of at least one sequence coding for a collagen peptide, but it can also encode additional peptide residues at the N- and / or C-terminal of the at least one collagen peptide, and so can the collagen peptide component can be a collagen fusion peptide.
- the nucleotide sequence coding for a collagen peptide is fused with at least one nucleotide sequence which codes for at least one, preferably hydrophilic, peptide residue.
- the fusion of the collagen peptide with the at least one peptide residue, preferably a hydrophilic peptide residue results in the formation of an overall hydrophobicity and / or proline-reduced collagen fusion peptide in the prokaryotic expression system.
- the collagen peptide component is a collagen fusion peptide which, in addition to the amino acid sequence of the collagen peptide, has at least one peptide residue, in particular a hydrophilic peptide residue, in particular at least has a protein tag, preferably His-tag, a signal peptide and / or a lamination domain.
- the at least one collagen peptide of the at least one collagen peptide component is preferably separated from the at least one peptide residue, in particular the at least one protein tag, the at least one signal peptide and / or the at least one lamination domain by specific recognition sequences.
- the specific recognition sequences are particularly preferably selected from the group consisting of factor Xa (Ile- (Glu / Asp) -Gly-Arg), TEV (Glu-Asn-Leu-Tyr-Phe-Gln- (Gly / Ser)), thrombin (Leu-Val-Pro-Arg-Gly-Ser), trypsin recognition sequence, papain recognition sequence.
- the lamination domain is an N-terminal amino acid sequence of the CollAl procollagen from Bos taurus or a V domain of the collagen-like protein ScI2.28 from Streptococcus pyogenes.
- the N-terminal amino acid sequence of the CollAl procollagen from Bos taurus preferably has the amino acid sequence according to SEQ ID no. 17, preferably consists of this.
- the V domain of the collagen-like protein ScI2.28 from Streptococcus pyogenes preferably has the amino acid sequence according to SEQ ID no. 18, preferably consists of this.
- the collagen peptide component according to the present invention can be fused with one or two peptide residues.
- the at least one peptide residue of the collagen peptide component in particular of the collagen fusion peptide, is maltose-binding protein (MBP).
- MBP is fused to the N-terminus of the collagen peptide.
- MBP is fused to the C-terminus of the collagen peptide.
- MBP preferably has the amino acid sequence according to SEQ ID no. 7, preferably consists of this.
- the at least one peptide residue of the collagen peptide component in particular the Collagen fusion peptide around Superfolder-Green-Fluorescent Protein (Superfolder-GFP).
- Superfolder GFP is preferably fused to the N-terminus of the collagen peptide.
- Superfolder-GFP is fused to the C-terminus of the collagen peptide.
- Superfolder-GFP preferably has the amino acid sequence according to SEQ ID no. 5, preferably consists of this.
- the at least one peptide residue of the collagen peptide component, in particular of the collagen fusion peptide is Mxe-GyrA-intein with a C-terminal chitin binding domain.
- Mxe-GyrA-Intein with a C-terminal chitin binding domain is preferably fused to the N-terminus of the collagen peptide.
- Mxe-GyrA-Intein with a C-terminal chitin binding domain is fused to the C-terminus of the collagen peptide.
- Mxe-GyrA-Intein with a C-terminal chitin binding domain preferably has the amino acid sequence according to SEQ ID NO. 8, preferably consists of this.
- the at least one peptide residue of the collagen peptide component, in particular of the collagen fusion peptide, is preferably Mxe-GyrA-intein with a C-terminal chitin binding domain and superfolder GFP.
- Mxe-GyrA-Intein with a C-terminal chitin binding domain and superfolder GFP is preferably fused to the N-terminus of the collagen peptide.
- Mxe-GyrA-Intein with a C-terminal chitin binding domain and superfolder GFP is fused to the C-terminus of the collagen peptide.
- Mxe-GyrA-Intein with C-terminal chitin binding domain and Superfolder-GFP preferably has the amino acid sequence according to SEQ ID no. 9, preferably consists of this.
- the collagen peptide of the collagen peptide component in particular the collagen fusion peptide, is fused at the N terminus with MBP and at the C terminus with superfolder GFP, Mxe-GyrA-intein with C-terminal chitin binding domain or Mxe -GyrA-intein with C-terminal chitin binding domain and Superfolder-GFP fused.
- the collagen peptide component in particular the collagen fusion peptide, comprises a collagen peptide and at least one N- and / or C-terminal secretion signal peptide, preferably a cleavable, in particular enzymatically cleavable, N- and / or C-terminal one Secretion signal peptide.
- the collagen peptide component in particular the collagen fusion peptide, a collagen peptide, an N- and / or C-terminal secretion signal peptide and at least one further peptide residue, in particular at least one further hydrophilic peptide residue.
- the N- and / or C-terminal secretion signal peptide is particularly preferably selected from HlyA, HlyAc and the catalytic domain of a cellulase from Bacillus subtilis KSM-64.
- the HlyA signal peptide sequence particularly preferably contains the amino acid sequence according to SEQ ID no. 1, preferably consists of this.
- the HlyAc signal peptide sequence contains the amino acid sequence according to SEQ ID no. 2, preferably consists of this.
- the collagen peptide component is particularly preferably a collagen fusion peptide in which the collagen peptide is fused at the N terminus and / or C terminus with a hydrophilic peptide residue, in particular with superfolder GFP at the N terminus and with an HlyA signal sequence or an HlyAc - Signal sequence at the C-terminus.
- the superfolder GFP preferably has the amino acid sequence according to SEQ ID no. 5, preferably consists of this.
- the collagen peptide component is particularly preferably a collagen fusion peptide in which the collagen peptide is fused at the N terminus with the catalytic domain of a cellulase from Bacillus subtilis KSM-64.
- the catalytic domain of a cellulase from Bacillus subtilis KSM-64 preferably has the amino acid sequence according to SEQ ID no. 6, preferably consists of this.
- the collagen peptide component in particular the collagen fusion peptide, can have further peptide residues fused to the N terminus and / or C terminus of the collagen peptide in addition to the catalytic domain of a cellulase from Bacillus subtilis KSM-64.
- the prokaryotic expression system provided in step a) additionally comprises at least one HlyB and at least one HlyD-coding nucleotide sequence and is cultured in step b) in the culture medium under conditions that the expression of the at least one recombinant collagen peptide component and of HlyB and Enable HlyD. Due to the co-expression of HlyB, HlyD and a collagen peptide component comprising the secretion signal peptide, it is advantageously possible to secrete the collagen peptide component into the culture medium.
- HlyB preferably has the amino acid sequence according to SEQ ID no. 3, preferably consists of this.
- HylD has the amino acid sequence according to SEQ ID no. 4, preferably consists of this.
- the collagen peptide of the collagen peptide component in particular the collagen fusion peptide
- the secretion signal peptide in particular with the signal sequence HlyA, the signal sequence HlyAc or with the catalytic domain of a cellulase from Bacillus subtilis KSM-64 according to the aforementioned embodiments of the present invention It is advantageously possible to form recombinant collagen peptide components, in particular collagen peptides or collagen fusion peptides, in a prokaryotic expression system and to secrete them directly into the culture medium.
- the collagen peptide component in particular the collagen fusion peptide, comprises a collagen peptide and at least one N-terminal signal peptide, preferably a cleavable, in particular enzymatically cleavable, N-terminal Sec- or TAT-specific signal peptide.
- the prokaryotic expression system is an E. coli leaky mutant.
- the collagen peptide component in particular the collagen peptide or the collagen fusion peptide, is present intracellularly and thus with a large number of host proteins.
- it is advantageously also no longer necessary to partially or completely disrupt the cells periplasmic expression: selective periplasmic disruption; cytosolic expression: complete lysis of the cell, which also avoids isolating the collagen peptide component from a complex protein mixture have to.
- the recombinant collagen peptide component is subject to proteolysis by intracellular proteases.
- the synthesized collagen peptide component By translocating the synthesized collagen peptide component into the periplasmic space of a leaky mutant or by direct secretion into the culture medium, its purification is considerably simplified and more economical.
- the collagen peptide component in the culture medium is largely protected from proteolysis.
- the secretion of the collagen peptide component into the culture medium often enables higher product titers to be achieved than with cytosolic expression. Since the collagen peptide component according to these preferred embodiments is largely free of host proteins in the culture medium, their isolation does not require affinity chromatography or multi-stage, complex purification, but only an ultra-
- the cell disruption process step can advantageously be dispensed with.
- the collagen peptide component in particular the collagen peptide or the
- Collagen fusion peptide a cleavage of C- and / or N-terminal procollagen fragments to obtain a collagen peptide component, in particular a collagen peptide or a collagen fusion peptide, take place.
- the collagen peptide component obtained with the method according to the invention in step b) and obtained in step c) has at least one fused to the N- and / or C-terminus of the collagen peptide, in particular cleavable, preferably enzymatically cleavable, Peptide or protein sequence.
- This at least one peptide or protein sequence fused to the N- and / or C-terminus of the collagen peptide reduces the proline content and / or the hydrophobicity of the collagen fusion peptide expressed in the prokaryotic expression system during expression in the prokaryotic expression system and thus affects the antimicrobial effectiveness of the against collagen peptides.
- the recombinant collagen peptide component encoded by the nucleotide sequence of the prokaryotic expression system to be formed under conditions which in process step b) post-transcriptionally, namely either i) post-transcriptional and pre-translational or ii) post-transcriptional and post-translational, to a reduction in the hydrophobicity and / or the proline content of the collagen peptide encoded by the nucleotide sequence Component with respect to the at least one of the collagen peptide components coding nucleotide sequence encoded collagen peptide lead.
- the recombinant collagen peptide component encoded by the nucleotide sequence of the prokaryotic expression system is formed under conditions in which post-transcriptional and pre-translational, i.e. before or during translation of the mRNA, at least a low hydrophobicity having amino acid instead of a hydrophobic amino acid provided by the base triplet of the mRNA, in particular proline, is incorporated into the collagen peptide component, in particular into the collagen peptide or the collagen fusion peptide.
- this can be done through the use of prokaryotic expression systems that do not produce proline, in particular no hydrophobic amino acid, but instead the lower hydrophobicity-exhibiting amino acid hydroxyproline and incorporate it into the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, by way of translation so that the hydrophobicity and / or the proline content of the collagen peptide component formed, in particular the collagen peptide or collagen fusion peptide, is reduced compared to the at least one collagen peptide encoded by the collagen peptide component-encoding nucleotide sequence.
- the hydrophobicity and / or the proline content of the collagen peptide component in particular the collagen peptide or the collagen fusion peptide, can be reduced by culturing the prokaryotic expression system in a hydroxyproline-containing or hydroxyproline-enriched culture medium in step b).
- the cultivation in step b) of the method according to the invention takes place in a hydroxyproline-containing or with hydroxyproline enriched culture medium.
- the hydroxyproline-containing or hydroxyproline-enriched culture medium is particularly preferably obtained by incubating a proline-containing or proline-enriched culture medium with at least one proline-4-hydroxylase (PIN4H).
- the prokaryotic expression system is a proline-auxotrophic host cell. In a further preferred embodiment it can be provided that the prokaryotic expression system is a proline-auxotrophic and hydroxyproline-producing host cell.
- the prokaryotic expression system is a proline-auxotrophic host cell and the culture medium is hydroxyproline-containing or hydroxyproline-enriched, in particular the hydroxyproline-containing or hydroxyproline-enriched culture medium by incubating a proline-containing or proline-enriched culture medium with at least one proline-4-hydroxylase (PIN4H).
- PIN4H proline-4-hydroxylase
- the reduction in the hydrophobicity and / or the proline content can be done by using a prokaryotic expression system which, in addition to the at least one nucleotide sequence coding for at least one recombinant collagen peptide component, also expresses at least one nucleotide sequence coding for at least one proline-4-hydroxylase.
- the reduction in the hydrophobicity and / or the proline content takes place post-transcriptionally and pre-translationally using the proline 4-hydroxylase (PIN4H) expressed by the prokaryotic system (EC 1.14.11.57).
- PIN4Hs convert the natural amino acid L-proline using oxygen and 2-oxoglutarate to L-hydroxyproline, which is recognized by the proline tRNA synthetase and instead of L-proline in a growing polypeptide chain of the collagen peptide component, in particular the collagen peptide or the Collagen fusion peptide.
- the present invention relates in particular to a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems, comprising the steps: i) Provision of a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one nucleotide sequence coding for at least one proline 4-hydroxylase (PIN4H), the nucleotide sequence coding for at least one recombinant collagen peptide component being the nucleotide sequence of at least one collagen peptide comprises, ii) cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one recombinant collagen peptide component and the at least one proline 4-hydroxylase (PIN4H) to obtain at least one hydroxylated collagen peptide component, the collagen peptide Component has a reduced hydrophobicity and / or a reduced proline content compared to the at least one
- Proline 4-hydroxylase of bacterial origin is particularly preferred, in particular proline 4-hydroxylase is a proline 4-hydroxylase from Streptomyces griseoviridis, Dactylosporangium sp., Pseudomonas stutzeri, Bordetella bronchiseptica, Bradyrhizobium japonicum, Aeromonas cobaviae, Janthin sp. or Achromobacter xylosoxidans.
- the proline-4-hydroxylase is a monomeric proline-4-hydroxylase.
- the proline 4-hydroxylase has an amino acid sequence selected from SEQ ID no. 10 to 13, preferably consists of an amino acid sequence selected from SEQ ID NO. 10 to 13.
- the proline 4-hydroxylase particularly preferably has the amino acid sequence according to SEQ ID no. 10, preferably consists of this.
- the proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 11, preferably consists of this.
- the proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 12, preferably consists of this.
- the proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 13, preferably consists of this.
- post-translational Modification in particular hydroxylations, in particular proline hydroxylations and / or glycosylations
- the prokaryotic expression system provided in step a) additionally comprises at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase and is cultivated in step b) in a culture medium under conditions that promote the expression of the at least one recombinant collagen peptide Component and the at least one prolyl 4-hydroxylase, the collagen peptide component having a reduced hydrophobicity and / or a reduced prolin content compared to the at least one nucleotide sequence encoded by the collagen peptide component.
- the prokaryotic expression system provided in step a) is preferably E. coli. In a further preferred embodiment of the present invention, the prokaryotic expression system provided in step a) is Bacillus subtilis.
- the at least one nucleotide sequence of the prokaryotic expression system coding for at least one recombinant collagen peptide component is preferably a codon-optimized nucleic acid, in particular at least one nucleotide sequence coding for the preferred codon use of the prokaryotic expression system .
- the method according to the invention advantageously enables a recombinant collagen peptide that is generally toxic to the prokaryotic expression system, in particular in E. coli, to be produced with high efficiency and in high purity.
- the procedure according to the invention is particularly advantageous in that recombinant collagen peptides can also be produced in an industrial or large-scale process.
- the recombinant collagen peptide components provided according to the invention are biologically active.
- the collagen peptide components produced with the method according to the invention show a biological effectiveness on the biosynthesis of proteins of the extracellular matrix, preferably on the biosynthesis of collagen, elastin and / or proteoglycans.
- the collagen peptide components produced with the method according to the invention particularly preferably show a biological activity on chondrocytes, fibroblasts and / or osteoblasts.
- the at least one nucleotide sequence coding for at least one recombinant collagen peptide component is particularly preferably codon-optimized.
- the at least one collagen peptide encoded by the at least one nucleotide sequence preferably has a collagen of types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII , XVIII, XIX, XX, XXI, XXII, XXIII, XIV, XXV, XXVI, XXVII, preferably type I, II or III, preferably type I, preferably type II, preferably type III.
- the at least one collagen peptide encoded by the at least one nucleotide sequence preferably has one in collagen from vertebrates, in particular fish, amphibians, reptiles, birds and mammals, in particular in human, bovine, porcine, equine or avian collagen of types I, II or III Type I, preferably type II, preferably type III, occurring amino acid sequence.
- the collagen peptide encoded by the nucleotide sequence particularly preferably has an amino acid sequence occurring in bovine collagen, in particular in bovine type I collagen, preferably in the a1 chain of bovine type I collagen.
- the collagen peptide in particular the collagen peptide of the collagen peptide component or the collagen peptide of the collagen fusion peptide, preferably has at least one amino acid sequence selected from the group consisting of SEQ ID NO. 25, 26, 27, 28, 29, 30, 31 and 33, preferably consists of at least one of these.
- the collagen peptide of the collagen fusion peptide encoded by the at least one nucleotide sequence is preferably a naturally occurring collagen peptide.
- the collagen peptide of the collagen fusion peptide encoded by the nucleotide sequence is not a naturally occurring collagen peptide.
- the collagen peptide of the collagen fusion peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide.
- this is through the nucleotide sequence
- Coded collagen peptide of the collagen fusion peptide is a genetically modified collagen peptide in which at least one amino acid of the amino acid sequence of a naturally occurring collagen peptide, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser, the amino acid sequence of a naturally occurring collagen peptide, is replaced by at least one very specific amino acid, in particular by at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, has been replaced.
- the recombinant collagen peptide component in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a size of 0.18 to 110 kDa, preferably 0.18 to 100 kDa, preferably 0.18 to 90 kDa, preferably 0.18 to 80 kDa, preferably 0.18 to 70 kDa, preferably 0.2 to 60 kDa, preferably 0.3 to 50 kDa , preferably 0.5 to 50 kDa, preferably 0.6 to 50 kDa, preferably 0.7 to 50 kDa, preferably 0.8 to 50 kDa, preferably 0.9 to 50 kDa, preferably 1 to 50 kDa, preferably 2 to 50 kDa, preferably 5 to 50 kDa, preferably 5 to 40 kDa,
- the recombinant collagen peptide component in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a size of 0.18 up to 20 kDa, preferably 0.2 to 18 kDa, preferably 0.3 to 16 kDa, preferably 0.5 to 14 kDa, preferably 0.6 to 12 kDa, preferably 0.8 to 10 kDa, preferably 1 to 8 kDa, preferably 1 to 6 kDa, preferably 1 to 4 kDa.
- the recombinant collagen peptide component in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, preferably has a size of 10 to 80 kDa 10 to 70 kDa, preferably 10 to 60 kDa, preferably 10 to 50 kDa, preferably 10 to 40 kDa, preferably 10 to 30 kDa, preferably 10 to 20 kDa.
- the recombinant collagen peptide component in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, is particularly preferably hydroxylated.
- the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue is not hydroxylated.
- the recombinant collagen peptide component in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a ratio of proline to hydroxyproline from 0% to 45% proline to 55% to 100% hydroxyproline (based on the number of proline and hydroxyproline residues of the collagen peptide component).
- the present invention also comprises a collagen peptide component which can be produced, in particular produced, by the method according to the invention for producing a recombinant collagen peptide component in prokaryotic systems.
- the present invention also relates to a method for producing a recombinant hydroxylated collagen peptide in prokaryotic systems, comprising the steps: aa) providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one collagen peptide and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase , bb) cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide, cc) obtaining at least one hydroxylated collagen peptide, the at least one Collagen peptide has the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
- the method according to the invention thus advantageously allows the production of at least one hydroxylated
- the at least one collagen peptide obtained in step cc) has a degree of hydroxylation of 5 to 100%, preferably 5 to 90%, preferably 5 to 80%, preferably 10 to 70%, preferably 15 to 60%, preferably 20 to 50%, preferably 30 to 50%, preferably 35 to 50%, preferably 40 to 50% (each based on the total number of proline and lysine residues of the collagen peptide).
- the at least one collagen peptide obtained in step cc) preferably has a degree of hydroxylation of at least 5%, preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50% (each based on the total number of proline and lysine residues of the collagen peptide).
- the at least one collagen peptide obtained in step cc) preferably has a degree of hydroxylation of at most 80%, preferably at most 75%, preferably at most 70%, preferably at most 65%, preferably at most 60%, preferably at most 55%, preferably at most 50% ( each based on the total number of proline and lysine residues of the collagen peptide).
- the nucleotide sequence coding for at least one collagen peptide is a naturally occurring nucleotide sequence.
- the nucleotide sequence coding for at least one collagen peptide is preferably a nucleotide sequence from mammals.
- the nucleotide sequence coding for at least one collagen peptide is preferably a nucleotide sequence of bovine origin.
- the nucleotide sequence preferably encodes a naturally occurring collagen peptide.
- the nucleotide sequence coding for at least one collagen peptide is not a natural one occurring nucleotide sequence.
- the nucleotide sequence coding for at least one collagen peptide is preferably a genetically modified nucleotide sequence.
- the nucleotide sequence does not encode a naturally occurring collagen peptide.
- the collagen peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide.
- the collagen peptide encoded by the nucleotide sequence is a genetically modified collagen peptide in which at least one amino acid of the amino acid sequence of a naturally occurring collagen peptide, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser , the amino acid sequence of a naturally occurring collagen peptide, is replaced by at least one very specific amino acid, in particular by at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp became.
- the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of bacterial or plant origin. At least one nucleotide sequence coding for prolyl-4-hydroxylase is preferably a nucleotide sequence of bacterial origin. In a further preferred embodiment of the present invention, the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of plant origin, preferably a nucleotide sequence from Arabidopsis thaliana. The at least one prolyl-4-hydroxylase-coding nucleotide sequence of plant origin, preferably from Arabidopsis thaliana, is preferably codon-optimized.
- the nucleotide sequence encoding at least one prolyl 4-hydroxylase preferably has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95 %, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the nucleotide sequence according to SEQ ID no. 14 on.
- the nucleotide sequence encoding at least one prolyl 4-hydroxylase particularly preferably encodes at least one prolyl 4-hydroxylase from Arabidopsis thaliana, in particular at least one prolyl 4-hydroxylase comprising an amino acid sequence according to SEQ ID no. 15th
- the prolyl-4-hydroxylase in particular the prolyl-4-hydroxylase from Arabidopsis thaliana
- the prolyl 4-hydroxylase in particular the prolyl 4-hydroxylase from Arabidopsis thaliana
- the at least one prolyl 4-hydroxylase, in particular the prolyl 4-hydroxylase from Arabidopsis thaliana has an amino acid sequence according to SEQ ID no. 16, preferably consists of this.
- the present invention also relates to a hydroxylated collagen peptide which can be produced, in particular produced, by the method according to the invention for producing a recombinant hydroxylated collagen peptide in prokaryotic systems.
- the present invention further comprises a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems, comprising the method for producing a recombinant collagen peptide component in prokaryotic systems according to the present invention, the prokaryotic expression system provided in step a) additionally comprises at least one nucleotide sequence coding for at least one prolyl 4-hydroxylase and the prokaryotic expression system in step b) is carried out in a culture medium under conditions which enable the expression of the at least one collagen peptide and the at least one prolyl 4-hydroxylase.
- the present invention in particular also comprises a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: i) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one at least one Nucleotide sequence coding for prolyl-4-hydroxylase, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, ii) culturing the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide Component and the at least one prolyl-4-hydroxylase enable to obtain at least one hydroxylated collagen peptide component, the collagen peptide component having a reduced hydrophobicity and / or a reduced prolin content compared to the at least one collagen
- the collagen peptide component obtained in step iii), in particular the collagen peptide or collagen fusion peptide has the amino acid sequence motif (Gly-XY) n , with at least 50% of the hydroxylations in the at least one collagen peptide on a proline in Y position are present.
- the present invention relates in particular to a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: a) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one at least one prolyl 4-hydroxylase-coding nucleotide sequence, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, b) cultivating the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component and the at least one Enable prolyl 4-hydroxylase to obtain at least one hydroxylated collagen peptide component, the collagen peptide component opposite the at least one of the collagen peptide -Component-encoding nucleotide sequence-encoded collagen peptide has a
- the at least one prolyl-4-hydroxylase encoded by the at least one nucleotide sequence is a prolyl-4-hydroxylase which has a specificity, in particular predominant specificity, for the hydroxylation of in the Y position of the amino acid sequence motif (Gly-XY) Has n lying proline residues.
- the preferred, in particular predominant, hydroxylation of proline residues of the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, in the Y position of the amino acid sequence motif (Gly-XY) n advantageously achieves a hydroxylation pattern that corresponds to the hydroxylation pattern of collagen and collagen peptides from vertebrates, in particular Fish, amphibians, reptiles, birds and mammals, in particular human, bovine, porcine, equine or avian collagen and collagen peptides, preferably bovine collagen and collagen peptides.
- the collagen peptide component in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n once, preferably twice, preferably three times, preferably four times.
- the collagen peptide component, in particular the collagen peptide or collagen fusion peptide preferably has the amino acid sequence motif (Gly-XY) n at least once, preferably at least twice, preferably at least three times, preferably at least four times.
- the collagen peptide component in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n at most twice, preferably at most three times, preferably at most four times.
- N is particularly preferably an integer> 1, preferably> 2, preferably> 3, preferably> 4, preferably> 5, preferably> 6, preferably> 7, preferably> 8, preferably> 9, preferably> 10, preferably> 15, preferably> 20, preferably> 25, preferably> 30, preferably> 35, preferably> 40, preferably> 45, preferably> 50.
- the amino acid sequence motif (Gly-XY) n in the amino acid sequence of the collagen peptide component, in particular the Collagen peptide or collagen fusion peptide occurs x times, for example once, twice, three times or four times, where n is an integer> 1, preferably> 2, preferably> 3, preferably> 4, preferably> 5, preferably> 6, preferably> 7 , preferably> 8, preferably> 9, preferably> 10, preferably> 15, preferably> 20, preferably> 25, preferably> 30, preferably> 35, preferably> 40, preferably> 45, preferably> 50.
- the present invention also relates to a hydroxylated collagen peptide component which can be produced, in particular produced, by one of the methods according to the invention for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems.
- the term “collagen” is understood in a manner customary in the art, in particular as defined, for example, in WO 01/34646.
- the term “collagen” relates to collagen types I to XXVII.
- the term “collagen” is understood to mean a peptide having the sequence glycine-proline, glycine-4-hydroxyproline or glycine-X-4-hydroxyproline, preferably the repetitive motif (Gly-XY) n, where X and Y can be any amino acid, preferably proline and 4-hydroxylproline.
- the term “collagen” is particularly preferably understood to mean a peptide having the repetitive motif (Gly-Pro-Y) n and / or (Gly-X-Hyp) m , where X and Y can be any amino acid.
- collagen peptide is understood to mean a protein or peptide which has an amino acid sequence occurring in collagen according to the above definition, the protein or peptide being at least one dipeptide, preferably an oligopeptide or polypeptide, acts.
- the collagen peptide can in particular be present in chemically modified form, in particular hydroxylated and / or glycosylated form, or it can be unmodified.
- a “collagen peptide” within the meaning of the present invention can also be a collagen protein.
- the collagen peptide of the present invention can be present in single-stranded form, but the collagen peptide of the present invention can also be present as a dimer or trimer, in particular a trimer, from the same or different collagen peptides, in particular also as a triple-helical collagen peptide.
- a “naturally occurring collagen peptide” is understood to mean a collagen peptide that can be isolated directly from natural sources, that is, one that has an amino acid sequence as encoded in naturally occurring nucleotide sequences of an organism, in particular without mutations in these nucleotide sequences Occurrence, especially those that lead to one or more amino acid exchanges.
- naturally occurring collagen peptides are understood to mean that they occur naturally in a vertebrate, in particular in cattle, or in an invertebrate, in particular a jellyfish.
- a naturally occurring collagen peptide is a collagen peptide which occurs in cattle.
- the term “collagen peptide component” denotes a peptide comprising at least the amino acid sequence of a collagen peptide.
- the collagen peptide component can consist of the amino acid sequence of the collagen peptide, that is to say it can be a collagen peptide.
- the collagen peptide component comprises the amino acid sequence of at least one collagen peptide and at least one further amino acid sequence that is not a collagen peptide.
- the collagen peptide component is a collagen fusion peptide.
- the collagen peptide component in particular the collagen fusion peptide, preferably has at least one peptide residue, in particular a hydrophilic peptide residue, fused N- and / or C-terminally to the amino acid sequence of the collagen peptide.
- a “host cell” is understood to mean a living cell which is capable of expressing peptides or proteins encoded in foreign DNA, in particular in recombinant DNA.
- a “recombinant collagen peptide” is a biotechnological recombinant production by means of a Expression system obtained collagen peptide understood. According to the invention, the “recombinant collagen peptide” is characteristic of not being obtained from natural sources.
- nucleotide sequence is understood to mean the sequence of the nucleotides of a nucleic acid, in particular a polynucleic acid strand, in particular a DNA strand.
- a “nucleotide sequence” is therefore to be understood both as an informational unit and as the DNA strand that physically manifests this information.
- the terms “codon optimization” and “codon optimized” are preferably used to adapt the nucleotide sequence, in particular the base triplets coding for an amino acid, to the expression system selected by the selected expression system, in particular the selected prokaryotic expression system Base triplets coding for a specific amino acid understood.
- the “codon optimization” is based on the fact that the different base triplets coding for a specific amino acid are used differently by different species in protein biosynthesis. Accordingly, a “codon optimization” leads to a change in the nucleic acid coding for a specific amino acid sequence, but not to a change in the coded amino acid sequence itself.
- a “reduction in the hydrophobicity” is understood to mean a reduction in the total hydrophobicity of the relevant collagen peptide component compared to the total hydrophobicity of the collagen peptide of the collagen peptide component.
- the “reduction in hydrophobicity” can be achieved, for example, by fusing the collagen peptide with at least one peptide residue, preferably with at least one hydrophilic peptide residue.
- the hydrophobicity of the collagen peptide component can be reduced, for example, by pre-translational or post-translational hydroxylation of proline to hydroxyproline.
- the hydrophobicity of the peptide can be determined, for example, using the Eisenberg Consensus Scale (ECS), the Gunnar von Heijne Scale, the Kyte and Doolittle Scale, the Wimley-White Scale, the aWW Scale, the Engelman-Steitz Scale or other determination methods known to the person skilled in the art .
- ECS Eisenberg Consensus Scale
- the reduction in the total hydrophobicity of the collagen peptide component in question compared to the total hydrophobicity of the collagen peptide of the collagen peptide component is preferably at least 5%, preferably at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%.
- the term “reduction in the proline content” denotes a reduction in the percentage of proline residues in the total number of amino acids in the relevant collagen peptide component compared to the percentage of proline residues in relation to the total number of amino acids in the at least one collagen peptide encoded by the collagen peptide component .
- a “reduction in the prolin content” of the collagen peptide component can be achieved, for example, in that the collagen peptide component is a collagen fusion peptide.
- the percentage of proline residues in the collagen peptide component is reduced compared to the percentage in the proline-rich collagen peptide.
- the reduction of the percentage of proline residues in the total number of amino acids of the relevant collagen peptide component compared to the percentage of proline residues in relation to the total number of amino acids in the at least one collagen peptide encoded by the collagen peptide component is preferably at least 5%, preferably at least 10 %, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%.
- the term “degree of hydroxylation” denotes the percentage of hydroxylated proline and lysine residues in the total number of proline and lysine residues of the collagen peptide component, in particular of the collagen peptide or the collagen fusion peptide.
- an “expression system” is understood to mean a system in which a targeted and controlled protein biosynthesis can take place.
- a “prokaryotic expression system” is to be understood according to the invention as a cell-based biological system which is able to carry out protein biosynthesis in a targeted and controlled manner, that is to say to synthesize peptides or proteins starting from a nucleic acid as an information carrier.
- the “expression” of a nucleotide sequence includes, in particular, the transcription into mRNA (messenger RNA) and the subsequent transcription Translation into a peptide or, synonymous in connection with the present invention, also protein.
- condition that enable the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component “conditions that enable the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide "," Conditions that enable the expression of the at least one recombinant collagen peptide component and the at least one prolyl 4-hydroxylase to obtain at least one hydroxylated collagen peptide component "and” Conditions that enable the expression of the at least one recombinant collagen peptide component and the enable at least one proline-4-hydroxylase (PIN4H) to obtain at least one hydroxylated collagen peptide component “according to the invention, conditions such as temperature, pressure, time, light and the presence or absence of inducers and / or repressors, are understood to mean that expression of the b Activate or amplify the relevant peptide and / or protein.
- the peptide and / or protein in question is expressed in the context of a high cell density fermentation, in particular under high pressure, preferably high pressure air.
- high pressure preferably high pressure air.
- the specific conditions which enable expression of the peptide and / or protein in question are known to the person skilled in the art and depend on the expression system used and the expression cassette used, in particular the promoter contained therein.
- the expression of the peptide and / or protein in question can be constitutive or inducible expression, depending on the structure of the expression cassette.
- obtaining a recombinant collagen fusion peptide refers to the isolation of a collagen peptide component, a collagen peptide or a collagen fusion peptide from a composition containing several components by means of known isolation processes, such as centrifugation processes, in particular differential centrifugation and / or density gradient centrifugation, chromatographic processes, in particular geifiltration, ion exchange, affinity and / or high-performance liquid chromatography, electrophoresis processes, filtration processes and / or extraction processes, where an enrichment and purification of the component in question from the several components containing composition can preferably be achieved by sequential application of several isolation methods.
- isolation processes such as centrifugation processes, in particular differential centrifugation and / or density gradient centrifugation, chromatographic processes, in particular geifiltration, ion exchange, affinity and / or high-performance liquid chromatography, electrophoresis processes, filtration processes and / or extraction processes, where an enrichment and purification of the component in question from the several
- the terms “comprising” and “having” are understood to mean that, in addition to the elements explicitly covered by these terms, further elements that are not explicitly mentioned can be added. In connection with the present invention, these terms are also understood to mean that only the explicitly mentioned elements are included and no further elements are present. In this particular embodiment, the meaning of the terms “comprising” and “having” is synonymous with the term “consisting of”. In addition, the terms “comprising” and “having” also include compositions which, in addition to the explicitly named elements, also contain other non-named elements, which are, however, of a functionally and qualitatively subordinate nature. In this embodiment, the terms “comprising” and “having” are synonymous with the term “consisting essentially of”.
- the first and second decimal places or the second decimal places are / is not specified, these are / is to be set as 0.
- the term “and / or” is understood to mean that all members of a group which are connected by the term “and / or” are disclosed both as alternatives to one another and also cumulatively with one another in any combination.
- A, B and / or C this means that the following disclosure content is to be understood: a) A or B or C or b) (A and B) or c) (A and C) or d) ( B and C) or e) (A and B and C).
- a method for producing a recombinant collagen peptide component in prokaryotic systems comprising the steps: a) Providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component, the nucleotide sequence coding for the at least one recombinant collagen peptide component the nucleotide sequence comprises at least one collagen peptide, b) Cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component, the collagen peptide component being compared to the at least one collagen peptide encoded by the collagen peptide component has a reduced hydrophobicity and / or a reduced proline content, c) recovering the collagen peptide component.
- Aspect 2 The method according to aspect 1, wherein in the nucleotide sequence of the prokaryotic expression system that codes for the collagen peptide component, the nucleotide sequence that codes for a collagen peptide is fused with at least one nucleotide sequence that codes for a peptide residue.
- Aspect 3 The method according to aspect 2, wherein the peptide residue is at least one protein tag
- Aspect 4 Method according to one of aspects 2 and 3, wherein the peptide residue can be split off, in particular can be split off enzymatically.
- Aspect 5 Method according to one of aspects 2 to 4, wherein after step c) or after step b) and before step c) the at least one peptide residue of the collagen peptide component is split off.
- Aspect 6 The method according to any one of aspects 2 to 5, wherein the collagen peptide component comprises a collagen peptide and at least one N- and / or C-terminal secretion signal peptide.
- Aspect 7 Method according to one of aspects 1 to 6, wherein the collagen peptide encoded by the nucleotide sequence has an amino acid sequence occurring in bovine collagen, in particular in bovine type I collagen, preferably in the a1 chain of bovine type I collagen.
- a method for producing a recombinant hydroxylated collagen peptide in prokaryotic systems comprising the steps: aa) providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one collagen peptide and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase, bb) Cultivating the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide, cc) obtaining at least one hydroxylated collagen peptide, the at least one collagen peptide having the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
- Aspect 9 The method according to aspect 8, wherein at least 80% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
- Aspect 10 The method according to aspect 8 or 9, wherein the at least one collagen peptide obtained in step cc) has a degree of hydroxylation of at least 5% (based on the total number of proline and lysine residues of the collagen peptide).
- Aspect 11 The method according to any one of aspects 8 to 10, wherein the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of bacterial or plant origin.
- Aspect 12 The method according to aspect 11, wherein the nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence from Arabidopsis thaliana.
- Aspect 13 Method for producing a recombinant hydroxylated collagen peptide
- prokaryotic expression system comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, ii ) Cultivating the prokaryoti expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide component, the collagen peptide component compared to the at least one collagen peptide encoded by the collagen peptide component-encoding nucleotide sequence has a reduced hydrophobicity and / or a reduced prolin content, iii) recovery
- Aspect 14 The method according to aspect 13, wherein the collagen peptide component obtained in step iii), in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide on a proline in Y Position are available.
- Aspect 15 Collagen peptide component produced by a method according to any one of Aspects 1 to 7.
- Aspect 16 Hydroxylated collagen peptide produced by a method according to any one of Aspects 8 to 12.
- Aspect 17 Hydroxylated collagen peptide component produced by the method according to any one of Aspects 13 and 14.
- Example 1 Cytosolic expression of a collagen peptide component in E. coli
- 1 pL of the vector pMAL-c5x (50-100 ng / pL) comprising a tetracycline resistance gene and one of SEQ ID no. 19 and 20, added to 50 pL thawed on ice, chemically competent E. coli BL21 and stored on ice for 30 min.
- the supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended.
- the cells are then streaked with 25 mg / L tetracycline on an LB agar plate preheated to 37 ° C. and incubated at 37 ° C. overnight.
- a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 25 mg / L tetracycline in a 300 mL shake flasks with baffles are used.
- the culture is cultivated overnight on an orbital shaker at 37 ° C. and 120 revolutions per minute (deflection 25 mm).
- the supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended.
- the cells are then spread on an LB agar plate preheated to 37 ° C. with 20 mg / L tetracycline and 20 mg / L chloramphenicol and incubated at 37 ° C. overnight.
- the supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended.
- the cells are then spread with 100 mg / L kanamycin on an LB agar plate preheated to 37 ° C. and incubated at 37 ° C. overnight.
- a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 100 mg / L Kanamycin in one 300 mL shake flask with baffles used.
- the culture is cultivated overnight on an orbital shaker at 37 ° C. and 120 revolutions per minute (deflection 25 mm).
- this previously set culture is not induced and cultivated overnight at 37 ° C. and used as the second stage preculture.
- the feed was fed (a total of 3 L volume consisting of 600 g / L glycerol, 90 g / L yeast extract, 2 g / L MgCU7H? C)).
- 12 standard liters of air per minute are used and a pressure of 0.2 bar is set.
- the pH is kept constant at 7.0 with 10% phosphoric acid and 4 M sodium hydroxide solution. Cultivation takes place at 37 ° C.
- OD600 9.0-10.0, 0.1 mM IPTG is induced.
- the cultivation is ended after the stationary phase has been reached.
- the vector pACYCDuet-1 comprising a chloramphenicol resistance gene and SEQ ID NO. 23, or the vector pCDFDuet-1, comprising a streptomycin resistance gene and SEQ ID no. 23, transformed into E. coli cells expressing a collagen peptide component.
- both the cytosolic expression can be obtained by co-expression with a preferably Y-position n hydroxylating prolyl hydroxylase 4-how of prolyl hydroxylase 4-1 from Arabidopsis thaliana, of hydroxylated collagen components (see. Example 1) and the secretory expression of hydroxylated collagen components (see. Examples 2 and 3) can be realized by E. coli cells.
- the post-translational hydroxylation of proline could be demonstrated by mass spectrometry.
- the determination of the degree of hydroxylation was based on the hydrolysis of the protein sample to be analyzed into the individual amino acids, their derivatization (AQC reagent) and the subsequent separation by means of analytical high-performance liquid chromatography (HPLC).
- the degree of hydroxylation was then calculated from the chromatogram with the aid of a proline and hydroxyproline standard from the peak areas of proline and hydroxyproline from the sample to be analyzed, the degree of hydroxylation being the proportion of hydroxylated proline residues (hydroxyproline) based on the molar sum of all proline (hydroxyproline and proline) is (mole hydroxyproline / (mole hydroxyproline + mole proline).
- the purified collagen peptides to be analyzed were hydrolyzed for> 24 h at 110 ° C. in 6 M hydrochloric acid (final concentration) with a protein load of 10 g / L in closed reaction vessels. After cooling on ice, neutralizing drop by drop on ice with sample-identical volume 6 M sodium hydroxide solution, centrifuging the samples for 5 min at 13,000 min 1 in a microliter centrifuge and, if necessary, diluting the samples with 0.2 M sodium borate buffer pH 9.0 the derivatization.
- AQC reagent 2 mg / mL 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate dissolved in acetonitrile (pA) at 55 ° C
- the 15 proline residues of the cellulase domain are not hydroxylated by the co-expressed At-P4H, because this part does not correspond to the natural At-P4H substrate spectrum, the actual degree of hydroxylation of the recombinant collagen sequence is higher than the degree of hydroxylation of the fusion protein.
- Pre-translational proline hydroxylation For the pre-translational hydroxylation of collagen peptide components, collagen peptide component exp dinating K. coli cells were combined with a vector comprising SEQ ID NO. 24 transformed in order to achieve a co-expression of collagen peptide components to be hydroxylated and a proline-4-hydroxylase (PIN4H) from Streptomyces griseoviridis. The incorporation of hydroxyproline into the expressed collagen peptide components could be demonstrated by mass spectroscopy.
- PIN4H proline-4-hydroxylase
- the degree of hydroxylation was determined as described in paragraph 1.1 above.
- the pre-translational hydroxylation approach using a proline-4-hydroxylase is unspecific, ie the incorporation of hydroxyproline instead of proline occurs randomly during translation and essentially depends on the availability of hydroxyproline -loaded tRNA. Since the co-expression of PIN4H and the collagen peptides only takes place after a sufficient protein-containing biomass has been built up during fermentation, it can be assumed that a certain proportion of host proteins, which may be present as contaminants in the protein sample to be analyzed, are present in non-hydroxylated form and the determined degree of hydroxylation is less than would be the case in the presence of a highly pure collagen peptide. It can therefore be assumed that the degrees of hydroxylation determined in the case of post-translational hydroxylation represent minimum values.
- Example 5 Cytosolic expression of a hydroxylated collagen peptide component in E. coli
- 1 pL of the vector pET-28a (+) - vector comprising a kanamycin resistance gene and a nucleic acid sequence according to SEQ ID no. 32, which contain the collagen peptide according to SEQ ID no. 33
- 1 pL of an expression plasmid which has either an At-P4H coding sequence (pACYCDuet-1) or an L # -RIN4H coding sequence (pCDFDuet-1) to 50 pL, chemically competent E. coli BL21, thawed on ice given and stored on ice for 30 min.
- the supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended.
- the cells are then spread on an LB agar plate preheated to 37 ° C. with 80 mg / L kanamycin and 20 mg / L chloramphenicol (P4H) or 20 mg / L streptomycin (PIN4H) and incubated at 37 ° C. overnight.
- the feed was added until the relative pCb value is below 60% again (a total of 3 L volume consisting of 600 g / L glycerol, 90 g / L yeast extract, 2 g / L MgCbUHiO). 15 standard liters of air per minute are used for gassing and an overpressure of 0.2 bar is set.
- the pH value is kept constant at 7.0 with 10% (w / w) phosphoric acid and 25% (w / w) ammonia water.
- Pluronic® PE 8100 is used as an anti-foaming agent. Cultivation takes place at 37 ° C. At an optical density of OD600 of 9.0-10.0, 0.1 mM IPTG is induced. The cultivation is ended after the stationary phase has been reached.
Abstract
The present invention relates to methods for producing hydroxylated collagen peptide components and to the hydroxylated collagen peptide components, in particular hydroxylated collagen peptides, obtained by this method.
Description
BESCHREIBUNG DESCRIPTION
Expression von Kollagenpeptid-Komponenten in prokaryotischen Systemen Expression of collagen peptide components in prokaryotic systems
Die vorliegende Erfindung betrifft Verfahren zur Herstellung hydroxylierter Kollagenpeptid- Komponenten sowie die aus diesen Verfahren erhaltenen hydroxylierten Kollagenpeptid- Komponenten, insbesondere hydroxylierten Kollagenpeptide. The present invention relates to processes for the production of hydroxylated collagen peptide components and the hydroxylated collagen peptide components obtained from these processes, in particular hydroxylated collagen peptides.
Kollagen ist das am häufigsten auftretende Protein im menschlichen Körper und verleiht unterschiedlichen Geweben als Hauptkomponente der extrazellulären Matrix die charakteristische Flexibilität und Elastizität. Kollagen-Polypeptidketten bilden eine Helix- Struktur aus, welche durch die repetitive Konsensus-Sequenz der Aminosäuren (Gly-X-Y)n hervorgerufen wird, wobei es sich bei „Gly“ für Glycin handelt und „X“ und „Y“ jeweils für eine beliebige Aminosäure stehen. Die am häufigsten auftretende repetitive Einheit in Kollagen ist Gly-Pro-Hyp, wobei „Pro“ für die Aminosäure Prolin steht und es sich bei „Hyp“ um Hydroxyprolin ((S)-(-)-irans-4- Hydroxyprolin) handelt. Der Hydroxylierungsgrad von Prolin in menschlichen Kollagenen variiert zwischen ca. 42-54 %. Bei Rinderkollagen liegt der Hydroxylierungsgrad im Bereich von 45 %. Collagen is the most common protein in the human body and, as the main component of the extracellular matrix, gives various tissues their characteristic flexibility and elasticity. Collagen polypeptide chains form a helix structure, which is caused by the repetitive consensus sequence of the amino acids (Gly-XY) n , where "Gly" is for glycine and "X" and "Y" are each for any one Amino acid stand. The most common repetitive unit in collagen is Gly-Pro-Hyp, where “Pro” stands for the amino acid proline and “Hyp” is hydroxyproline ((S) - (-) - irans-4-hydroxyproline). The degree of hydroxylation of proline in human collagens varies between approx. 42-54%. In the case of bovine collagen, the degree of hydroxylation is in the region of 45%.
Kollagen ist eine wichtige Quelle für biologisch aktive Peptide mit vielfältigen Anwendungsgebieten. Aufgrund ihrer hohen antioxidativen und antihypertensiven Aktivität in Kombination mit ihrer geringen Antigenität eignen sich Kollagenpeptide besonders für die Anwendung als „Functional Food“. Unter dem Aspekt der Nachhaltigkeit ist es erstrebenswert Kollagenpeptide insbesondere aus Säugern wie dem Rind ( Bos taurus ) rekombinant herzustellen. Für die rekombinante Produktion von Kollagen-Polypeptidketten tierischen, insbesondere bovinen, Ursprungs, bevorzugt Peptide oder Fragmente aus der natürlichen Sequenz einer Kollagen-Polypeptidkette, insbesondere des a-1-Typ-I-Kollagens (CollAl), kommen prinzipiell unterschiedliche zellbasierte Expressionssysteme in Frage: Prokaryoten, wie Escherichia coli ( E . coli ), sowie eukaryotische Organismen, wie Hefen, Pflanzen, Säugerzellen und Insektenzellen. Zwar besitzen Bakterien und Hefen keine natürlichen Mechanismen zur Hydroxylierung von Prolin, ermöglichen jedoch eine kosteneffiziente Produktion von rekombinanten Proteinen. Collagen is an important source of biologically active peptides with a wide range of applications. Due to their high antioxidant and antihypertensive activity in combination with their low antigenicity, collagen peptides are particularly suitable for use as "functional food". In terms of sustainability, it is desirable to recombinantly produce collagen peptides, especially from mammals such as cattle (Bos taurus). For the recombinant production of collagen polypeptide chains of animal, in particular bovine, origin, preferably peptides or fragments from the natural sequence of a collagen polypeptide chain, in particular α-1 type I collagen (CollAl), different cell-based expression systems are possible in principle : Prokaryotes such as Escherichia coli (E. coli) and eukaryotic organisms such as yeast, plants, mammalian cells and insect cells. Although bacteria and yeasts do not have natural mechanisms for hydroxylating proline, they enable the cost-efficient production of recombinant proteins.
Die Verwendung von prokaryoti sehen Systemen, wie E. coli , für die industrielle Produktion von rekombinanten Kollagenpeptiden bietet den Vorteil, dass diese vielfältig charakterisiert sind,
genetisch einfach zugänglich sind, eine geringe Komplexität besitzen, über eine hohe spezifische Wachstumsgeschwindigkeit verfügen, zu hohen Zelldichten angezogen werden können (Hochzelldichtefermentation), auf kostengünstigen Kulturmedien wachsen, eine schnelle Expression von rekombinanten Proteinen mit hohem Expressionsniveau ermöglichen und in prokaryoti sehen Systemen häufig hohe Raum-Zeit-Ausbeuten erzielt werden können. The use of prokaryotic systems, such as E. coli, for the industrial production of recombinant collagen peptides offers the advantage that these are characterized in many ways, are genetically easily accessible, have a low complexity, have a high specific growth rate, can be attracted to high cell densities (high cell density fermentation), grow on inexpensive culture media, enable rapid expression of recombinant proteins with a high level of expression and often see high space in prokaryotic systems -Time yields can be achieved.
Die rekombinante Expression der menschlichen a-Kette des Typ III Kollagens (hCol3Al) unter Einsatz des Wirtsorganismus Escherichia coli ist aus dem Stand der Technik bekannt (Shi et al., 2017, 36, 322-331; Rutschmann et al., Appl. Microbiol. Biotechnol. , 2014, 98, 4445- 4455). Trotz des charakteristischen Kollagen-Sequenzmotivs (Gly-X-Y)n weisen verschiedene Kollagene, insbesondere Kollagene unterschiedlichen Ursprungs, teils deutlich unterschiedliche Sequenzen und damit verbunden unterschiedliche Eigenschaften in Bezug auf die rekombinante Expression in prokaryoti sehen Systemen auf. So besitzt das bovine Coli Al (bCollAl) nur eine Sequenzidentität von 62,1 % und eine Sequenzähnlichkeit von 67,5 % zum humanen Col3Al, woraus sich ein anderes Verhalten hinsichtlich der Expression von bovinem Kollagen Coli Al ergibt und sich der im Stand der Technik beschriebene cytosolische Expressionsansatz auf die rekombinante Expression des bovinen Kollagens Coli Al in prokaryoti sehen Systemen nicht übertragen lässt. Die Produktion von rekombinanten Kollagenpeptiden, insbesondere Kollagenpeptiden bovinen Ursprungs, mit Hilfe prokaryoti scher Systeme, beispielsweise mit dem Wirtsorganismus Escherichia coli , stellt eine besondere Herausforderung dar, da Kollagenpeptide neben der gewünschten biologischen Aktivität auch antimikrobielle Wirkung besitzen können. Einige natürlich vorkommende Kollagenpeptide, wie beispielsweise das bovine CollAl, weisen im Vergleich zu anderen im Stand der Technik in E. coli bereits erfolgreich exprimierten Kollagenen - wie marine Kollagene, Kollagen-ähnlichen Proteinen (collagen-like proteins) bakteriellen oder künstlichen Ursprungs sowie sogenannten Designer-Kollagenen, beispielsweise bestehend aus repetitiven GEK (G: Glycin, E: Glutaminsäure, K: Lysin) und GDK-Abfolgen (D: Asparaginsäure) und nicht natürlich vorkommende Aminosäureabfolgen (keine 100 %-ige Übereinstimmung zu natürlichem Kollagen wie CollAl) - eine höhere Hydrophobizität (33-43 %) und/oder einen höheren Prolingehalt (20-31 %) auf. Aufgrund der dadurch bewirkten antimikrobiellen Wirkung erweist sich die rekombinante Expression vieler Kollagenpeptide in prokaryoti sehen Systemen daher als problematisch. The recombinant expression of the human α-chain of type III collagen (hCol3Al) using the host organism Escherichia coli is known from the prior art (Shi et al., 2017, 36, 322-331; Rutschmann et al., Appl. Microbiol . Biotechnol., 2014, 98, 4445- 4455). Despite the characteristic collagen sequence motif (Gly-XY) n , different collagens, in particular collagens of different origins, sometimes have significantly different sequences and, associated therewith, different properties with regard to recombinant expression in prokaryotic systems. The bovine Coli Al (bCollAl) only has a sequence identity of 62.1% and a sequence similarity of 67.5% to the human Col3Al, which results in a different behavior with regard to the expression of bovine collagen Coli Al and that in the prior art described cytosolic expression approach to the recombinant expression of the bovine collagen Coli Al in prokaryotic systems cannot be transferred. The production of recombinant collagen peptides, in particular collagen peptides of bovine origin, with the help of prokaryotic systems, for example with the host organism Escherichia coli, is a particular challenge, since collagen peptides can also have antimicrobial effects in addition to the desired biological activity. Some naturally occurring collagen peptides, such as bovine CollAl, have already successfully expressed collagens in E. coli in comparison to other prior art - such as marine collagens, collagen-like proteins of bacterial or artificial origin and so-called designers -Collagens, for example consisting of repetitive GEK (G: glycine, E: glutamic acid, K: lysine) and GDK sequences (D: aspartic acid) and non-naturally occurring amino acid sequences (no 100% match to natural collagen such as CollAl) - one higher hydrophobicity (33-43%) and / or a higher proline content (20-31%). Because of the antimicrobial effect brought about by this, the recombinant expression of many collagen peptides in prokaryotic systems therefore proves to be problematic.
Darüber hinaus besteht ein besonderes Problem in Hinblick auf die Herstellung rekombinanter hydroxylierter Kollagenpeptide darin, dass Prokaryoten im Unterschied zu vielen Eukaryoten
natürlicherweise weder post-translational noch prä-translational zur Hydroxylierung von Prolin befähigt sind. Aus dem Stand der Technik sind bereits verschiedene Ansätze bekannt, den Wirtsorganismus E. coli zur post-translationalen Hydroxylierung von (S)-Prolin (L-Prolin) unter Verwendung von Sauerstoff, 2-Oxoglutarat und Ascorbat zu (2S,4R)-4-Hydroxyprolin (L-Hydroxyprolin) zu befähigen. So wurde die rekombinante Expression der humanen Prolyl-4- hydroxalse (P4H), bei welcher es sich um ein a2ß2-Tetramer handelt, bereits in aktiver Form sowohl im Cytosol, als auch im Periplasma von E. coli beschrieben (Kersteen et al., Protein Expr. Purij. ’, 2004, 38, 279-291; Neubauer et al Matrix Biol., 2005, 24 , 59-68). Darüber hinaus konnte die humane Prolyl-4-hydroxylase erfolgreich mit künstlichen Kollagensequenzen sowie einem human-ähnlichen Kollagen im Cytosol von E. coli co-exprimiert werden (Pinkas et al., ACS Chem. Biol., 2011, 6, 320-324; Tang et al., Appl. Biochem. Biotechnol. 2016, 178, 1458-1470). Wenngleich die humane P4H in E. coli in aktiver Form exprimiert werden konnte, liegen keinerlei Berichte über die rekombinante Expression der bovinen Prolyl-4-hydroxylase (P4H) in E. coli vor. Zu berücksichtigen ist hierbei, dass die bovine P4H nur eine geringe Homologie zur humanen P4H besitzt. Die a-Untereinheit aus dem Rind weißt eine Sequenzidentität von ca. 37 % und eine Sequenzähnlichkeit von ca. 57 % zur humanen a-Untereinheit auf und die ß-Untereinheit aus dem Rind besitzt eine Sequenzidentität von ca. 33 % und eine Sequenzähnlichkeit von ca. 50 % zur humanen ß-Untereinheit. Problematisch bei der rekombinanten Expression aller P4Hs aus Vertebraten in Prokaryoten ist, dass ausschließlich das a2ß2-Tetramer mit einem vergleichsweise sehr hohen Molekulargewicht von etwa 240 kDa katalytische Aktivität besitzt und für die Ausbildung der nativen Struktur der a-Untereinheit und die Assoziation zum Tetramer die Ausbildung intramolekularer Disulfidbrücken notwendig ist. Eine in vitro Assoziation der Untereinheiten zum Tetramer funktioniert nicht und die Co-expression der ß-Untereinheit ist notwendig, um die a-Untereinheit in löslicher Form zu halten. Ein weiteres Problem ist, dass die Aktivität und Stabilität des Tetramers stark von der Verfügbarkeit von Kollagensubstraten abhängt, was den Produktionsprozess und die Expressions-/Induktionsstrategie sehr schwierig gestaltet. Aufgrund der multimeren Organisation und der begrenzten Stabilität der tierischen Prolyl-4-hydroxylasen, gepaart mit deren sehr begrenzter Aktivität gegenüber kurzen Kollagensubstraten, ist eine effiziente und korrekte Hydroxylierung von Kollagenpeptiden, insbesondere von bovinen CollAl-Kollagenpeptiden, unter Einsatz der bovinen Prolyl-4- hydroxylase in E. coli nicht gewährleistet. Da zudem die Produktivität gewöhnlich mit steigender Anzahl an verschiedenen rekombinant zu exprimierenden Genen abnimmt, wäre eine monomere P4H mit einer Präferenz für die Kollagen-typische Hydroxylierung von Prolin in Y-Position wünschenswert. Bakterielle P4Hs werden zwar in verschiedenen Genomen vorhergesagt,
allerdings sind bisher nur sehr wenige bakterielle P4Hs charakterisiert worden, insbesondere hinsichtlich ihres Hydroxylierungsmusters. Ein Beispiel hierfür stellt die P4H aus Bacillus anthracis dar, welche allerdings sowohl in der X als auch in der Y-Position der Kollagenmotivs (Gly-X-Y)n hydroxyliert (Schnicker et al., ./. Biol. Chem ., 2016, 291, 13360-13374) und somit zur Erzeugung eines zum bovinen Coli Al möglichst identischen Hydroxylierungsmuster nicht in Frage kommt. In addition, a particular problem with regard to the production of recombinant hydroxylated collagen peptides is that prokaryotes, in contrast to many eukaryotes are naturally not capable of hydroxylating proline either post-translationally or pre-translationally. Various approaches are already known from the prior art to convert the host organism E. coli to post-translational hydroxylation of (S) -proline (L-proline) using oxygen, 2-oxoglutarate and ascorbate to (2S, 4R) -4 -Hydroxyproline (L-Hydroxyproline) to empower. The recombinant expression of human prolyl-4-hydroxalse (P4H), which is an a2ß2 tetramer, has already been described in active form both in the cytosol and in the periplasm of E. coli (Kersteen et al., Protein Expr. Purij. ' , 2004, 38, 279-291; Neubauer et al Matrix Biol., 2005, 24, 59-68). In addition, the human prolyl 4-hydroxylase could be successfully co-expressed with artificial collagen sequences and a human-like collagen in the cytosol of E. coli (Pinkas et al., ACS Chem. Biol., 2011, 6, 320-324; Tang et al., Appl. Biochem. Biotechnol. 2016, 178, 1458-1470). Although the human P4H could be expressed in E. coli in an active form, there are no reports of the recombinant expression of the bovine prolyl-4-hydroxylase (P4H) in E. coli. It should be noted here that bovine P4H has only a low degree of homology to human P4H. The bovine α subunit has a sequence identity of approx. 37% and a sequence similarity of approx. 57% to the human a subunit and the bovine β subunit has a sequence identity of approx. 33% and a sequence similarity of approx 50% to the human ß-subunit. The problem with the recombinant expression of all P4Hs from vertebrates in prokaryotes is that only the a2ß2 tetramer with a comparatively very high molecular weight of about 240 kDa has catalytic activity and the formation of the native structure of the a subunit and the association with the tetramer intramolecular disulfide bridges is necessary. An in vitro association of the subunits to the tetramer does not work and the co-expression of the ß-subunit is necessary to keep the a-subunit in soluble form. Another problem is that the activity and stability of the tetramer depends heavily on the availability of collagen substrates, which makes the production process and the expression / induction strategy very difficult. Due to the multimeric organization and the limited stability of the animal prolyl-4-hydroxylases, coupled with their very limited activity towards short collagen substrates, efficient and correct hydroxylation of collagen peptides, in particular of bovine CollAl collagen peptides, using the bovine prolyl-4 hydroxylase not guaranteed in E. coli. Since, in addition, productivity usually decreases with an increasing number of different genes to be expressed recombinantly, a monomeric P4H with a preference for the collagen-typical hydroxylation of proline in the Y position would be desirable. Bacterial P4Hs are predicted in different genomes, however, so far only very few bacterial P4Hs have been characterized, especially with regard to their hydroxylation pattern. An example of this is the P4H from Bacillus anthracis, which, however, is hydroxylated in both the X and Y positions of the collagen motif (Gly-XY) n (Schnicker et al., ./. Biol. Chem., 2016, 291 , 13360-13374) and is therefore out of the question for generating a hydroxylation pattern which is as identical as possible to the bovine Coli Al.
Der vorliegenden Erfindung liegt daher das technische Problem zugrunde ein Verfahren zur Herstellung einer rekombinant hergestellten hydroxylierten Kollagenpeptide-Komponente, insbesondere rekombinante hydroxylierte Kollagenpeptide, bereitzustellen, das die vorgenannten Nachteile überwindet, das es insbesondere erlaubt rekombinante hydroxylierte Kollagenpeptid- Komponenten, insbesondere rekombinante hydroxylierte Kollagenpeptide, auch im größerem industriellen und kostengünstigen Maßstab, in prokaryoti sehen Systemen herzustellen. Der vorliegenden Erfindung liegt insbesondere das technische Problem zugrunde ein Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente mit Kollagen- typischem Hydroxylierungsmuster in prokaryoti sehen Systemen, bereitzustellen. The present invention is therefore based on the technical problem of providing a method for producing a recombinantly produced hydroxylated collagen peptide component, in particular recombinant hydroxylated collagen peptides, which overcomes the aforementioned disadvantages, which in particular allows recombinant hydroxylated collagen peptide components, in particular recombinant hydroxylated collagen peptides, too on a larger industrial and cost-effective scale, see systems in prokaryoti. The present invention is based, in particular, on the technical problem of providing a method for producing a recombinant hydroxylated collagen peptide component with a collagen-typical hydroxylation pattern in prokaryotic systems.
Die vorliegende Erfindung löst das ihr zugrundeliegende technische Problem durch den Gegenstand der unabhängigen Ansprüche, insbesondere durch die Bereitstellung von Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente gemäß der vorliegenden Erfindung. The present invention solves the technical problem on which it is based by means of the subject matter of the independent claims, in particular by providing methods for producing a recombinant hydroxylated collagen peptide component according to the present invention.
Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung einer rekombinanten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend die Schritte: a) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, b) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente zum Erhalt mindestens einer Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid-Komponente gegenüber dem mindestens einen von der Kollagenpeptid-
Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, c) Gewinnen der Kollagenpeptid-Komponente. The present invention relates to a method for producing a recombinant collagen peptide component in prokaryotic see systems, comprising the steps: a) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence encoding at least one recombinant collagen peptide component, the at least one recombinant collagen peptide component coding nucleotide sequence comprises the nucleotide sequence of at least one collagen peptide, b) culturing the prokaryotic see expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component, the collagen peptide component compared to the at least one from the collagen peptide Component-encoding nucleotide sequence-encoded collagen peptide has a reduced hydrophobicity and / or a reduced prolin content, c) recovery of the collagen peptide component.
Die vorliegende Erfindung basiert insbesondere auf der Identifizierung der Kombination aus dem Prolingehalt und der Hydrophobizität als entscheidende Einflussfaktoren auf die antimikrobielle Wirksamkeit einiger Kollagenpeptide. Dabei führt der hydrophobe Charakter der Kollagenpeptide vermutlich zur Einlagerung der Peptide in die bakteriellen Membranen und damit zum Verlust der Membranintegrität, so wie es bei vielen extrazellulär applizierten antimikrobiellen Peptiden der Fall ist. Der hohe Prolingehalt der Kollagenpeptide führt vermutlich in Analogie zur Klasse der prolinreichen antimikrobiellen Peptide zu einer Störung des Zellmetabolismus auf Ebene der Proteinsynthese ohne Störung der Zellmembran. Das erfindungsgemäße Verfahren geht von einem prokaryoti sehen Expressionssystem, bevorzugt E. coli , aus, das mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz aufweist, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, das heißt dass die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz zumindest aus einer für ein Kollagenpeptid kodierende Sequenz besteht, jedoch von dieser auch zusätzliche Peptidreste N- und/oder C-terminal des mindestens einen Kollagenpeptids kodiert sein können, es sich bei der Kollagenpeptid-Komponente also auch um ein Kollagenfusionspeptid handeln kann. The present invention is based in particular on the identification of the combination of the proline content and the hydrophobicity as decisive influencing factors on the antimicrobial effectiveness of some collagen peptides. The hydrophobic character of the collagen peptides presumably leads to the storage of the peptides in the bacterial membranes and thus to the loss of membrane integrity, as is the case with many extracellularly applied antimicrobial peptides. In analogy to the class of proline-rich antimicrobial peptides, the high prolin content of the collagen peptides presumably leads to a disruption of cell metabolism at the level of protein synthesis without disrupting the cell membrane. The method according to the invention is based on a prokaryotic expression system, preferably E. coli, which has at least one nucleotide sequence coding for at least one recombinant collagen peptide component, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, that is to say that the nucleotide sequence coding for at least one recombinant collagen peptide component consists of at least one sequence coding for a collagen peptide, but it can also encode additional peptide residues at the N- and / or C-terminal of the at least one collagen peptide, and so can the collagen peptide component can be a collagen fusion peptide.
In einer bevorzugten Ausführungsform ist in der die Kollagenpeptid-Komponente kodierenden Nukleotidsequenz des prokaryoti sehen Expressionssystems die ein Kollagenpeptid kodierende Nukleotidsequenz mit mindestens einer Nukleotidsequenz fusioniert, die für mindestens einen, vorzugsweise hydrophilen, Peptidrest kodiert. Durch die Fusion des Kollagenpeptids mit dem mindestens einen Peptidrest, bevorzugt hydrophile Peptidrest, kommt es in dem prokaryoti sehen Expressionssystem zur Bildung eines insgesamt Hydrophobizitäts- und/oder Prolin-reduzierten Kollagenfusionspeptids. In a preferred embodiment, in the nucleotide sequence of the prokaryotic expression system coding for the collagen peptide component, the nucleotide sequence coding for a collagen peptide is fused with at least one nucleotide sequence which codes for at least one, preferably hydrophilic, peptide residue. The fusion of the collagen peptide with the at least one peptide residue, preferably a hydrophilic peptide residue, results in the formation of an overall hydrophobicity and / or proline-reduced collagen fusion peptide in the prokaryotic expression system.
Gemäß dieser bevorzugten Ausführungsform ist die Kollagenpeptid-Komponente ein Kollagenfusionspeptid, welches zusätzlich zu der Aminosäuresequenz des Kollagenpeptids mindestens einen Peptidrest, insbesondere ein hydrophiler Peptidrest, insbesondere mindestens
einen Protein-tag, bevorzugt His-tag, ein Signalpeptid und/oder eine Kaschierungsdomäne aufweist. According to this preferred embodiment, the collagen peptide component is a collagen fusion peptide which, in addition to the amino acid sequence of the collagen peptide, has at least one peptide residue, in particular a hydrophilic peptide residue, in particular at least has a protein tag, preferably His-tag, a signal peptide and / or a lamination domain.
Bevorzugt ist das mindestens eine Kollagenpeptid der mindestens einen Kollagenpeptid- Komponente, insbesondere des Kollagenfusionspeptids, von dem mindestens einen Peptidrest, insbesondere dem mindestens einen Protein-tag, dem mindestens einen Signalpeptid und/oder der mindestens einer Kaschierungsdomäne durch spezifische Erkennungssequenzen getrennt. The at least one collagen peptide of the at least one collagen peptide component, in particular the collagen fusion peptide, is preferably separated from the at least one peptide residue, in particular the at least one protein tag, the at least one signal peptide and / or the at least one lamination domain by specific recognition sequences.
Besonders bevorzugt sind die spezifischen Erkennungssequenzen ausgewählt aus der Gruppe bestehend aus Faktor Xa (Ile-(Glu/Asp)-Gly-Arg), TEV (Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)), Thrombin (Leu-Val-Pro-Arg-Gly-Ser), Trypsin-Erkennungssequenz, Papain-Erkennungssequenz. The specific recognition sequences are particularly preferably selected from the group consisting of factor Xa (Ile- (Glu / Asp) -Gly-Arg), TEV (Glu-Asn-Leu-Tyr-Phe-Gln- (Gly / Ser)), thrombin (Leu-Val-Pro-Arg-Gly-Ser), trypsin recognition sequence, papain recognition sequence.
In einer weiteren bevorzugten Ausführungsform ist die Kaschierungsdomäne eine N-terminale Aminosäuresequenz des CollAl-Prokollagen aus Bos taurus oder eine V-Domäne des Kollagen- ähnlichen Proteins ScI2.28 aus Streptococcus pyogenes. Bevorzugt weist die N-terminale Aminosäuresequenz des CollAl-Prokollagens aus Bos taurus die Aminosäuresequenz gemäß SEQ ID No. 17 auf, bevorzugt besteht aus dieser. Bevorzugt weist die V-Domäne des Kollagen- ähnlichen Proteins ScI2.28 aus Streptococcus pyogenes die Aminosäuresequenz gemäß SEQ ID No. 18 auf, bevorzugt besteht aus dieser. In a further preferred embodiment, the lamination domain is an N-terminal amino acid sequence of the CollAl procollagen from Bos taurus or a V domain of the collagen-like protein ScI2.28 from Streptococcus pyogenes. The N-terminal amino acid sequence of the CollAl procollagen from Bos taurus preferably has the amino acid sequence according to SEQ ID no. 17, preferably consists of this. The V domain of the collagen-like protein ScI2.28 from Streptococcus pyogenes preferably has the amino acid sequence according to SEQ ID no. 18, preferably consists of this.
In einer besonders bevorzugten Ausführungsform kann die Kollagenpeptid-Komponente gemäß der vorliegenden Erfindung mit einem oder mit zwei Peptidresten fusioniert sein. Bevorzugt ist das Kollagenpeptid der die Kollagenpeptid-Komponente mit mindestens einem, bevorzugt mindestens zwei, bevorzugt mindestens drei, bevorzugt mindestens vier, Peptidresten, insbesondere hydrophilen Peptidresten, fusioniert. In a particularly preferred embodiment, the collagen peptide component according to the present invention can be fused with one or two peptide residues. The collagen peptide which fuses the collagen peptide component with at least one, preferably at least two, preferably at least three, preferably at least four, peptide residues, in particular hydrophilic peptide residues, is preferred.
In besonders bevorzugter Ausführungsform der vorliegenden Erfindung handelt es sich bei dem mindestens einen Peptidrest der Kollagenpeptidkomponente, insbesondere des Kollagenfusionspeptids, um Maltose-bindende Protein (MBP). Bevorzugt ist MBP an den N- Terminus des Kollagenpeptids fusioniert. In einer weiteren bevorzugten Ausführungsform ist MBP an den C-Terminus des Kollagenpeptids fusioniert. Bevorzugt weist MBP die Aminosäuresequenz gemäß SEQ ID No. 7 auf, bevorzugt besteht aus dieser. In a particularly preferred embodiment of the present invention, the at least one peptide residue of the collagen peptide component, in particular of the collagen fusion peptide, is maltose-binding protein (MBP). Preferably, MBP is fused to the N-terminus of the collagen peptide. In a further preferred embodiment, MBP is fused to the C-terminus of the collagen peptide. MBP preferably has the amino acid sequence according to SEQ ID no. 7, preferably consists of this.
Gemäß einer bevorzugten Ausführungsform der vorliegenden Erfindung handelt es sich bei dem mindestens einen Peptidrest der Kollagenpeptid-Komponente, insbesondere des
Kollagenfusionspeptids um Superfolder-Grün-Fluoreszierendes Protein (Superfolder-GFP). Bevorzugt ist Superfolder-GFP an den N-Terminus des Kollagenpeptids fusioniert. In einer weiteren bevorzugten Ausführungsform ist Superfolder-GFP an den C-Terminus des Kollagenpeptids fusioniert. Bevorzugt weist Superfolder-GFP die Aminosäuresequenz gemäß SEQ ID No. 5 auf, bevorzugt besteht aus dieser. According to a preferred embodiment of the present invention, the at least one peptide residue of the collagen peptide component, in particular the Collagen fusion peptide around Superfolder-Green-Fluorescent Protein (Superfolder-GFP). Superfolder GFP is preferably fused to the N-terminus of the collagen peptide. In a further preferred embodiment, Superfolder-GFP is fused to the C-terminus of the collagen peptide. Superfolder-GFP preferably has the amino acid sequence according to SEQ ID no. 5, preferably consists of this.
In einer weiteren bevorzugten Ausführungsform handelt es sich bei dem mindestens einen Peptidrest der Kollagenpeptid-Komponente, insbesondere des Kollagenfusionspeptids, um Mxe- GyrA-Intein mit C-terminaler Chitinbinde-Domäne. Bevorzugt ist Mxe-GyrA-Intein mit C- terminaler Chitinbinde-Domäne an den N-Terminus des Kollagenpeptids fusioniert. In einer weiteren bevorzugten Ausführungsform ist Mxe-GyrA-Intein mit C-terminaler Chitinbinde- Domäne an den C-Terminus des Kollagenpeptids fusioniert. Bevorzugt weist Mxe-GyrA-Intein mit C-terminaler Chitinbinde-Domäne die Aminosäuresequenz gemäß SEQ ID No. 8 auf, bevorzugt besteht aus dieser. In a further preferred embodiment, the at least one peptide residue of the collagen peptide component, in particular of the collagen fusion peptide, is Mxe-GyrA-intein with a C-terminal chitin binding domain. Mxe-GyrA-Intein with a C-terminal chitin binding domain is preferably fused to the N-terminus of the collagen peptide. In a further preferred embodiment, Mxe-GyrA-Intein with a C-terminal chitin binding domain is fused to the C-terminus of the collagen peptide. Mxe-GyrA-Intein with a C-terminal chitin binding domain preferably has the amino acid sequence according to SEQ ID NO. 8, preferably consists of this.
Bevorzugt handelt es sich bei dem mindestens einen Peptidrest der Kollagenpeptid-Komponente, insbesondere des Kollagenfusionspeptids, um Mxe-GyrA-Intein mit C-terminaler Chitinbinde- Domäne und Superfolder-GFP. Bevorzugt ist Mxe-GyrA-Intein mit C-terminaler Chitinbinde- Domäne und Superfolder-GFP an den N-Terminus des Kollagenpeptids fusioniert. In einer weiteren bevorzugten Ausführungsform ist Mxe-GyrA-Intein mit C-terminaler Chitinbinde- Domäne und Superfolder-GFP an den C-Terminus des Kollagenpeptids fusioniert. Bevorzugt weist Mxe-GyrA-Intein mit C-terminaler Chitinbinde-Domäne und Superfolder-GFP die Aminosäuresequenz gemäß SEQ ID No. 9 auf, bevorzugt besteht aus dieser. The at least one peptide residue of the collagen peptide component, in particular of the collagen fusion peptide, is preferably Mxe-GyrA-intein with a C-terminal chitin binding domain and superfolder GFP. Mxe-GyrA-Intein with a C-terminal chitin binding domain and superfolder GFP is preferably fused to the N-terminus of the collagen peptide. In a further preferred embodiment, Mxe-GyrA-Intein with a C-terminal chitin binding domain and superfolder GFP is fused to the C-terminus of the collagen peptide. Mxe-GyrA-Intein with C-terminal chitin binding domain and Superfolder-GFP preferably has the amino acid sequence according to SEQ ID no. 9, preferably consists of this.
In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung ist das Kollagenpeptid der Kollagenpeptid-Komponente, insbesondere des Kollagenfusionspeptids, am N-Terminus mitMBP fusioniert und am C-Terminus mit Superfolder-GFP, Mxe-GyrA-Intein mit C-terminaler Chitinbinde-Domäne oder Mxe-GyrA-Intein mit C-terminaler Chitinbinde-Domäne und Superfolder-GFP fusioniert. In a particularly preferred embodiment of the present invention, the collagen peptide of the collagen peptide component, in particular the collagen fusion peptide, is fused at the N terminus with MBP and at the C terminus with superfolder GFP, Mxe-GyrA-intein with C-terminal chitin binding domain or Mxe -GyrA-intein with C-terminal chitin binding domain and Superfolder-GFP fused.
In einer bevorzugten Ausführungsform der vorliegenden Erfindung umfasst die Kollagenpeptid- Komponente, insbesondere das Kollagenfusionspeptid, ein Kollagenpeptid und mindestens ein N- und/oder C-terminales Sekretions-Signalpeptid, bevorzugt ein abspaltbares, insbesondere enzymatisch abspaltbares, N- und/oder C-terminales Sekretions-Signalpeptid. Bevorzugt umfasst
die Kollagenpeptid-Komponente, insbesondere das Kollagenfusionspeptid, ein Kollagenpeptid, ein N- und/oder C-terminales Sekretions-Signalpeptid und mindestens einen weiteren Peptidrest, insbesondere mindestens einen weiteren hydrophilen Peptidrest. Besonders bevorzugt ist das N- und/oder C-terminales Sekretions-Signalpeptid ausgewählt aus HlyA, HlyAc und der katalytischen Domäne einer Cellulase aus Bacillus subtilis KSM-64. In a preferred embodiment of the present invention, the collagen peptide component, in particular the collagen fusion peptide, comprises a collagen peptide and at least one N- and / or C-terminal secretion signal peptide, preferably a cleavable, in particular enzymatically cleavable, N- and / or C-terminal one Secretion signal peptide. Preferably includes the collagen peptide component, in particular the collagen fusion peptide, a collagen peptide, an N- and / or C-terminal secretion signal peptide and at least one further peptide residue, in particular at least one further hydrophilic peptide residue. The N- and / or C-terminal secretion signal peptide is particularly preferably selected from HlyA, HlyAc and the catalytic domain of a cellulase from Bacillus subtilis KSM-64.
Besonders bevorzugt enthält die HlyA-Signalpeptidsequenz die Aminosäuresequenz gemäß SEQ ID No. 1, bevorzugt besteht aus dieser. The HlyA signal peptide sequence particularly preferably contains the amino acid sequence according to SEQ ID no. 1, preferably consists of this.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung enthält die HlyAc- Signalpeptidsequenz die Aminosäuresequenz gemäß SEQ ID No. 2, bevorzugt besteht aus dieser. In a further preferred embodiment of the present invention, the HlyAc signal peptide sequence contains the amino acid sequence according to SEQ ID no. 2, preferably consists of this.
Besonders bevorzugt ist die Kollagenpeptid-Komponente ein Kollagenfusionspeptid, bei dem das Kollagenpeptid am N-Terminus und/oder C-Terminus mit einem hydrophilen Peptidrest, fusioniert ist, insbesondere mit Superfolder-GFP am N-Terminus und mit einer HlyA- Signalsequenz oder einer HlyAc- Signal Sequenz am C-Terminus. The collagen peptide component is particularly preferably a collagen fusion peptide in which the collagen peptide is fused at the N terminus and / or C terminus with a hydrophilic peptide residue, in particular with superfolder GFP at the N terminus and with an HlyA signal sequence or an HlyAc - Signal sequence at the C-terminus.
Bevorzugt weist das Superfolder-GFP die Aminosäuresequenz gemäß SEQ ID No. 5 auf, bevorzugt besteht aus dieser. The superfolder GFP preferably has the amino acid sequence according to SEQ ID no. 5, preferably consists of this.
Besonders bevorzugt ist die Kollagenpeptid-Komponente ein Kollagenfusionspeptid, bei dem das Kollagenpeptid am N-Terminus mit der katalytischen Domäne einer Cellulase aus Bacillus subtilis KSM-64 fusioniert ist. Bevorzugt weist die katalytische Domäne einer Cellulase aus Bacillus subtilis KSM-64 die Aminosäuresequenz gemäß SEQ ID No. 6 auf, bevorzugt besteht aus dieser. In dieser bevorzugten Ausführungsform kann die Kollagenpeptid-Komponente, insbesondere das Kollagenfusionspeptid, neben der katalytischen Domäne einer Cellulase aus Bacillus subtilis KSM-64 noch weitere mit dem N-Terminus und/oder C-Terminus des Kollagenpeptids fusionierte Peptidreste aufweisen. The collagen peptide component is particularly preferably a collagen fusion peptide in which the collagen peptide is fused at the N terminus with the catalytic domain of a cellulase from Bacillus subtilis KSM-64. The catalytic domain of a cellulase from Bacillus subtilis KSM-64 preferably has the amino acid sequence according to SEQ ID no. 6, preferably consists of this. In this preferred embodiment, the collagen peptide component, in particular the collagen fusion peptide, can have further peptide residues fused to the N terminus and / or C terminus of the collagen peptide in addition to the catalytic domain of a cellulase from Bacillus subtilis KSM-64.
In einer bevorzugten Ausführungsform umfasst das in Schritt a) bereitgestellte prokaryotische Expressionssystem zusätzlich mindestens eine HlyB und mindestens eine HlyD kodierende Nukleotidsequenz und wird in Schritt b) in dem Kulturmedium unter Bedingungen kultiviert, die die Expression der mindestens einen rekombinanten Kollagenpeptid-Komponente und von HlyB und HlyD ermöglichen. Durch die Co-Expression von HlyB, HlyD und einer das Sekretions- Signalpeptid umfassenden Kollagenpeptid-Komponente ist er vorteilhafterweise möglich, die Kollagenpeptid-Komponente in das Kulturmedium zu sezernieren.
Bevorzugt weist HlyB die Aminosäuresequenz gemäß SEQ ID No. 3 auf, bevorzugt besteht aus dieser. In einer weiteren bevorzugten Ausführungsform weist HylD die Aminosäuresequenz gemäß SEQ ID No. 4 auf, bevorzugt besteht aus dieser. In a preferred embodiment, the prokaryotic expression system provided in step a) additionally comprises at least one HlyB and at least one HlyD-coding nucleotide sequence and is cultured in step b) in the culture medium under conditions that the expression of the at least one recombinant collagen peptide component and of HlyB and Enable HlyD. Due to the co-expression of HlyB, HlyD and a collagen peptide component comprising the secretion signal peptide, it is advantageously possible to secrete the collagen peptide component into the culture medium. HlyB preferably has the amino acid sequence according to SEQ ID no. 3, preferably consists of this. In a further preferred embodiment, HylD has the amino acid sequence according to SEQ ID no. 4, preferably consists of this.
Durch die Fusion des Kollagenpeptids der Kollagenpeptid-Komponente, insbesondere des Kollagenfusionspeptids, mit dem Sekretions-Signalpeptid, insbesondere mit der Signalsequenz HlyA, der Signalsequenz HlyAc oder mit der katalytischen Domäne einer Cellulase aus Bacillus subtilis KSM-64 gemäß den vorgenannten Ausführungsformen der vorliegenden Erfindung ist es vorteilhafterweise möglich, rekombinante Kollagenpeptid-Komponenten, insbesondere Kollagenpeptide oder Kollagenfusionspeptide, in einem prokaryoti sehen Expressionssystem zu bilden und diese unmittelbar in das Kulturmedium zu sezemieren. By fusing the collagen peptide of the collagen peptide component, in particular the collagen fusion peptide, with the secretion signal peptide, in particular with the signal sequence HlyA, the signal sequence HlyAc or with the catalytic domain of a cellulase from Bacillus subtilis KSM-64 according to the aforementioned embodiments of the present invention It is advantageously possible to form recombinant collagen peptide components, in particular collagen peptides or collagen fusion peptides, in a prokaryotic expression system and to secrete them directly into the culture medium.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung umfasst die Kollagenpeptid-Komponente, insbesondere das Kollagenfusionspeptid, ein Kollagenpeptid und mindestens ein N-terminales Signalpeptid, bevorzugt ein abspaltbares, insbesondere enzymatisch abspaltbares, N-terminales Sec- oder TAT-spezifisches Signalpeptid. In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung handelt es sich bei dem prokaryoti sehen Expressionssystem um eine E.coli leaky -Mutante. Durch die über das N-terminale Sec- oder TAT-spezifische Signalpeptid vermittelte Translokation der Kollagenpeptid- Komponente, insbesondere des Kollagenpeptids oder des Kollagenfusionspeptids, in den periplasmatischen Raum ist es mit einer E. coli leaky -Mutante vorteilhafterweise möglich, die in den periplasmatischen Raum translozierte Kollagenpeptid-Komponente, insbesondere das Kollagenfusionspeptid, über die teildurchlässige Außenmembran der leaky-Mutante in das Kulturmedium zu sekretieren. In a further preferred embodiment of the present invention, the collagen peptide component, in particular the collagen fusion peptide, comprises a collagen peptide and at least one N-terminal signal peptide, preferably a cleavable, in particular enzymatically cleavable, N-terminal Sec- or TAT-specific signal peptide. In a particularly preferred embodiment of the present invention, the prokaryotic expression system is an E. coli leaky mutant. Through the translocation of the collagen peptide component, in particular the collagen peptide or the collagen fusion peptide, mediated via the N-terminal Sec- or TAT-specific signal peptide, into the periplasmic space, it is advantageously possible with an E. coli leaky mutant to enter the periplasmic space to secrete translocated collagen peptide component, in particular the collagen fusion peptide, via the partially permeable outer membrane of the leaky mutant into the culture medium.
Bei den vorgenannten Ausführungsformen der vorliegenden Erfindung kann vorteilhafterweise vermieden werden, die Kollagenpeptid-Komponente aus dem Cytoplasma oder aus einem periplasmatischen Raum des prokaryoti sehen Systems isolieren zu müssen. Insbesondere kann so vermieden werden, dass die Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder das Kollagenfusionspeptid, intrazellulär und somit mit einer Vielzahl von Wirtsproteinen vorliegt. Gemäß diesen bevorzugten Ausführungsformen ist es vorteilhafterweise auch nicht mehr notwendig, die Zellen partiell oder vollständig aufzuschließen (periplasmatische Expression: selektiver Periplasma-Aufschluss; cytosolische Expression: vollständige Lyse der Zelle) wodurch auch vermieden wird, die Kollagenpeptid-Komponente aus einer komplexen Proteinmischung isolieren zu müssen. Insbesondere bei Expression im Cytoplasma besteht die Gefahr, dass die
rekombinante Kollagenpeptid-Komponente einer Proteolyse durch intrazelluläre Proteasen unterliegt. Durch die Translokation der synthetisierten Kollagenpeptid-Komponente in den periplasmatischen Raum einer leaky-Mutante oder durch direkte Sekretion in das Kulturmedium wird deren Aufreinigung erheblich vereinfacht und ökonomischer. Gleichzeitig ist die Kollagenpeptid-Komponente im Kulturmedium weitestgehend vor Proteolyse geschützt. Darüber hinaus können durch die Sekretion der Kollagenpeptid-Komponente in das Kulturmedium oftmals höhere Produkttiter erzielt werden als bei einer cytosolischen Expression. Da die Kollagenpeptid- Komponente gemäß dieser bevorzugten Ausführungsformen im Kulturmedium weitestgehend frei von Wirtsproteinen vorliegt, bedarf es zu deren Isolierung keiner affmitätschromatographi sehen oder mehrstufigen, komplexen Aufreinigung, sondern lediglich eines Ultra-In the aforementioned embodiments of the present invention, it is advantageously possible to avoid having to isolate the collagen peptide component from the cytoplasm or from a periplasmic space of the prokaryotic system. In particular, it can thus be avoided that the collagen peptide component, in particular the collagen peptide or the collagen fusion peptide, is present intracellularly and thus with a large number of host proteins. According to these preferred embodiments, it is advantageously also no longer necessary to partially or completely disrupt the cells (periplasmic expression: selective periplasmic disruption; cytosolic expression: complete lysis of the cell), which also avoids isolating the collagen peptide component from a complex protein mixture have to. In particular with expression in the cytoplasm, there is a risk that the recombinant collagen peptide component is subject to proteolysis by intracellular proteases. By translocating the synthesized collagen peptide component into the periplasmic space of a leaky mutant or by direct secretion into the culture medium, its purification is considerably simplified and more economical. At the same time, the collagen peptide component in the culture medium is largely protected from proteolysis. In addition, the secretion of the collagen peptide component into the culture medium often enables higher product titers to be achieved than with cytosolic expression. Since the collagen peptide component according to these preferred embodiments is largely free of host proteins in the culture medium, their isolation does not require affinity chromatography or multi-stage, complex purification, but only an ultra-
/Diafiltrationsschrittes. Des Weiteren kann vorteilhafterweise auf den Prozessschritt des Zellaufschlusses verzichtet werden. Gegebenenfalls kann vor, nach oder während der Gewinnung der Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder des/ Diafiltration step. Furthermore, the cell disruption process step can advantageously be dispensed with. Optionally, before, after or during the recovery of the collagen peptide component, in particular the collagen peptide or the
Kollagenfusionspeptids, eine Abspaltung von C- und/oder N-terminalen Prokollagenfragmenten zur Gewinnung von einer Kollagenpeptid-Komponente, insbesondere einem Kollagenpeptid oder einem Kollagenfusionspeptid, erfolgen. Collagen fusion peptide, a cleavage of C- and / or N-terminal procollagen fragments to obtain a collagen peptide component, in particular a collagen peptide or a collagen fusion peptide, take place.
In einer weiteren Ausführungsform ist vorgesehen, dass im Anschluss an Verfahrensschritt b) und vor Verfahrensschritt c) oder im Anschluss an Verfahrensschritt c) in einem Verfahrensschritt d) eine Abspaltung, insbesondere enzymatische Abspaltung, des mindestens einen Peptidrests von der Kollagenpeptid-Komponente, insbesondere dem rekombinanten Kollagenfusionspeptid, erfolgt. Gemäß dieser bevorzugten Ausführungsform der vorliegenden Erfindung weist die mit dem erfindungsgemäßen Verfahren in Schritt b) erhaltene und in Schritt c) gewonnene Kollagenpeptid-Komponente mindestens eine an den N- und/oder C-Terminus des Kollagenpeptids fusionierte, insbesondere abspaltbare, bevorzugt enzymatisch abspaltbare, Peptid- oder Proteinsequenz auf. Diese mindestens eine an den N- und/oder C-Terminus des Kollagenpeptids fusionierte Peptid- oder Proteinsequenz verringert den Prolingehalt und/oder die Hydrophobizität des in dem prokaryoti sehen Expressionssystem exprimierten Kollagenfusionspeptids während der Expression im prokaryoti sehen Expressionssystem und wirkt somit der antimikrobiellen Wirksamkeit des Kollagenpeptids entgegen. Gemäß dieser Ausführungsform erfolgt nach Schritt b) und vor Schritt c) oder nach dem Gewinnen der Kollagenpeptid-Komponente in Schritt c) bevorzugt eine Abspaltung, insbesondere enzymatische Abspaltung, der mindestens einen an den N- und/oder C-Terminus des Kollagenpeptids fusionierten Peptidsequenz, um schließlich ein isoliertes Kollagenpeptid zu erhalten.
Gemäß einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung kann zusätzlich oder alternativ zum Vorliegen eines N- und/oder C-terminal an das Kollagenpeptid fusionierten Peptidrests auch vorgesehen sein, dass die von der Nukleotidsequenz des prokaryoti sehen Expressionssystems kodierte rekombinante Kollagenpeptid-Komponente unter Bedingungen gebildet wird, die in Verfahrensschritt b) post-transkriptional, und zwar entweder i) post- transkriptional und prä-translational oder ii) post-transkriptional und post-translational, zu einer Reduzierung der Hydrophobizität und/oder des Prolingehalts der von der Nukleotidsequenz kodierten Kollagenpeptid-Komponente gegenüber dem mindestens einen von der Kollagenpeptid- Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid führen. In a further embodiment it is provided that after method step b) and before method step c) or after method step c) in a method step d) a cleavage, in particular enzymatic cleavage, of the at least one peptide residue from the collagen peptide component, in particular the recombinant collagen fusion peptide. According to this preferred embodiment of the present invention, the collagen peptide component obtained with the method according to the invention in step b) and obtained in step c) has at least one fused to the N- and / or C-terminus of the collagen peptide, in particular cleavable, preferably enzymatically cleavable, Peptide or protein sequence. This at least one peptide or protein sequence fused to the N- and / or C-terminus of the collagen peptide reduces the proline content and / or the hydrophobicity of the collagen fusion peptide expressed in the prokaryotic expression system during expression in the prokaryotic expression system and thus affects the antimicrobial effectiveness of the Against collagen peptides. According to this embodiment, after step b) and before step c) or after recovering the collagen peptide component in step c) there is preferably a cleavage, in particular enzymatic cleavage, of the at least one peptide sequence fused to the N- and / or C-terminus of the collagen peptide to finally obtain an isolated collagen peptide. According to a further preferred embodiment of the present invention, in addition or as an alternative to the presence of a peptide residue fused N- and / or C-terminally to the collagen peptide, provision can also be made for the recombinant collagen peptide component encoded by the nucleotide sequence of the prokaryotic expression system to be formed under conditions which in process step b) post-transcriptionally, namely either i) post-transcriptional and pre-translational or ii) post-transcriptional and post-translational, to a reduction in the hydrophobicity and / or the proline content of the collagen peptide encoded by the nucleotide sequence Component with respect to the at least one of the collagen peptide components coding nucleotide sequence encoded collagen peptide lead.
Besonders bevorzugt wird in Schritt b) die von der Nukleotidsequenz des prokaryoti sehen Expressionssystems kodierte rekombinante Kollagenpeptid-Komponente unter Bedingungen gebildet, bei denen post-transkriptional und prä-translational, das heißt vor der Translation oder während der Translation der mRNA, mindestens eine niedrige Hydrophobizität aufweisende Aminosäure an Stelle einer durch das Basentriplett der mRNA vorgesehene hydrophoben Aminosäure, insbesondere Prolin, in die Kollagenpeptid-Komponente, insbesondere in das Kollagenpeptid oder das Kollagenfusionspeptid, eingebaut wird. Particularly preferably, in step b) the recombinant collagen peptide component encoded by the nucleotide sequence of the prokaryotic expression system is formed under conditions in which post-transcriptional and pre-translational, i.e. before or during translation of the mRNA, at least a low hydrophobicity having amino acid instead of a hydrophobic amino acid provided by the base triplet of the mRNA, in particular proline, is incorporated into the collagen peptide component, in particular into the collagen peptide or the collagen fusion peptide.
In bevorzugter Ausführungsform kann dies durch die Verwendung solcher prokaryoti sehen Expressionssysteme geschehen, die kein Prolin, insbesondere keine hydrophobe Aminosäure, sondern stattdessen die geringere Hydrophobizität aufweisende Aminosäure Hydroxyprolin hersteilen und im Wege der Translation in die Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, einbauen, sodass die Hydrophobizität und/oder der Prolingehalt der gebildeten Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder Kollagenfusionspeptids, gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid verringert wird. In a preferred embodiment, this can be done through the use of prokaryotic expression systems that do not produce proline, in particular no hydrophobic amino acid, but instead the lower hydrophobicity-exhibiting amino acid hydroxyproline and incorporate it into the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, by way of translation so that the hydrophobicity and / or the proline content of the collagen peptide component formed, in particular the collagen peptide or collagen fusion peptide, is reduced compared to the at least one collagen peptide encoded by the collagen peptide component-encoding nucleotide sequence.
In einer besonders bevorzugten Ausführungsform kann eine Verringerung der Hydrophobizität und/oder des Prolingehalts der Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder des Kollagenfusionspeptids, durch Kultivieren des prokaryoti sehen Expressionssystems in einem Hydroxyprolin-haltigen oder mit Hydroxyprolin angereicherten Kulturmedium in Schritt b) erfolgen. Gemäß dieser bevorzugten Ausführungsform erfolgt die Kultivierung in Schritt b) des erfindungsgemäßen Verfahrens in einem Hydroxyprolin-haltigen oder mit Hydroxyprolin-
angereicherten Kulturmedium. Besonders bevorzugt wird das Hydroxyprolin-haltige oder mit Hydroxyprolin angereicherte Kulturmedium durch Inkubation eines Prolin-haltigen oder mit Prolin angereicherten Kulturmediums mit mindestens einer Prolin-4-hydroxylase (PIN4H) erhalten. In a particularly preferred embodiment, the hydrophobicity and / or the proline content of the collagen peptide component, in particular the collagen peptide or the collagen fusion peptide, can be reduced by culturing the prokaryotic expression system in a hydroxyproline-containing or hydroxyproline-enriched culture medium in step b). According to this preferred embodiment, the cultivation in step b) of the method according to the invention takes place in a hydroxyproline-containing or with hydroxyproline enriched culture medium. The hydroxyproline-containing or hydroxyproline-enriched culture medium is particularly preferably obtained by incubating a proline-containing or proline-enriched culture medium with at least one proline-4-hydroxylase (PIN4H).
In einer bevorzugten Ausführungsform der vorliegenden Erfindung kann vorgesehen sein, dass es sich bei dem prokaryoti sehen Expressionssystem um eine Prolin-auxotrophe Wirtszelle handelt. In einer weiteren bevorzugten Ausführungsform kann vorgesehen sein, dass es sich bei dem prokaryoti sehen Expressionssystem um eine Prolin-auxotrophe und Hydroxyprolin-herstellende Wirtszelle handelt. In a preferred embodiment of the present invention it can be provided that the prokaryotic expression system is a proline-auxotrophic host cell. In a further preferred embodiment it can be provided that the prokaryotic expression system is a proline-auxotrophic and hydroxyproline-producing host cell.
In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung kann vorgesehen sein, dass es sich bei dem prokaryoti sehe Expressionssystem um eine Prolin-auxotrophe Wirtszelle handelt und das Kulturmedium Hydroxyprolin-haltig oder Hydroxyprolin-angereichert ist, insbesondere wobei das Hydroxyprolin-haltige oder mit Hydroxyprolin angereicherte Kulturmedium durch Inkubation eines Prolin-haltigen oder mit Prolin angereicherten Kulturmediums mit mindestens einer Prolin-4-hydroxylase (PIN4H) erhalten wird. In a particularly preferred embodiment of the present invention it can be provided that the prokaryotic expression system is a proline-auxotrophic host cell and the culture medium is hydroxyproline-containing or hydroxyproline-enriched, in particular the hydroxyproline-containing or hydroxyproline-enriched culture medium by incubating a proline-containing or proline-enriched culture medium with at least one proline-4-hydroxylase (PIN4H).
Erfindungsgemäß kann die Verringerung der Hydrophobizität und/oder des Prolingehalts durch die Verwendung eines prokaryoti sehen Expressionssystems geschehen, das neben der mindestens einen mindestens eine rekombinante Kollagenpeptid-Komponente kodierenden Nukleotidsequenz zusätzlich auch mindestens eine mindestens eine Prolin-4-hydroxylase kodierende Nukleotidsequenz exprimiert. Demnach erfolgt die Verringerung der Hydrophobizität und/oder des Prolingehalts post-transkriptional und prä-translational unter Einsatz der vom prokaryoti sehen System exprimierten Prolin-4-hydroxylase (PIN4H) (EC 1.14.11.57). PIN4Hs setzen die natürliche Aminosäure L-Prolin unter Verwendung von Sauerstoff und 2-Oxoglutarat zu L-Hydroxyprolin um, welches von der Prolin-tRNA-Synthetase erkannt und statt L-Prolin in eine wachsende Polypeptidkette der Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder des Kollagenfusionspeptids, eingebaut wird. According to the invention, the reduction in the hydrophobicity and / or the proline content can be done by using a prokaryotic expression system which, in addition to the at least one nucleotide sequence coding for at least one recombinant collagen peptide component, also expresses at least one nucleotide sequence coding for at least one proline-4-hydroxylase. Accordingly, the reduction in the hydrophobicity and / or the proline content takes place post-transcriptionally and pre-translationally using the proline 4-hydroxylase (PIN4H) expressed by the prokaryotic system (EC 1.14.11.57). PIN4Hs convert the natural amino acid L-proline using oxygen and 2-oxoglutarate to L-hydroxyproline, which is recognized by the proline tRNA synthetase and instead of L-proline in a growing polypeptide chain of the collagen peptide component, in particular the collagen peptide or the Collagen fusion peptide.
Folglich betrifft die vorliegende Erfindung insbesondere ein Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend die Schritte:
i) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolin-4-hydroxylase (PIN4H) kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, ii) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolin-4-hydroxylase (PIN4H) zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, iii) Gewinnen der hydroxylierten Kollagenpeptid-Komponente. Accordingly, the present invention relates in particular to a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems, comprising the steps: i) Provision of a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one nucleotide sequence coding for at least one proline 4-hydroxylase (PIN4H), the nucleotide sequence coding for at least one recombinant collagen peptide component being the nucleotide sequence of at least one collagen peptide comprises, ii) cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one recombinant collagen peptide component and the at least one proline 4-hydroxylase (PIN4H) to obtain at least one hydroxylated collagen peptide component, the collagen peptide Component has a reduced hydrophobicity and / or a reduced proline content compared to the at least one nucleotide sequence encoded by the collagen peptide component, iii) recovering the hydroxyl ated collagen peptide component.
Besonders bevorzugt ist die Prolin-4-hydroxylase bakteriellen Ursprungs, insbesondere ist die Prolin-4-hydroxylase eine Prolin-4-hydroxylase aus Streptomyces griseoviridis, Dactylosporangium sp., Pseudomonas stutzeri, Bordetella bronchiseptica, Bradyrhizobium japonicum, Aeromonas caviae , Janthinobacterium sp. oder Achromobacter xylosoxidans. In einer besonders bevorzugten Ausführungsform handelt es sich bei der Prolin-4-hydroxylase um eine monomere Prolin-4-hydroxylase. In einer weiteren bevorzugten Ausführungsform weist die Prolin-4-hydroxylase eine Aminosäuresequenz ausgewählt aus SEQ ID No. 10 bis 13 auf, bevorzugt besteht aus einer Aminosäuresequenz ausgewählt aus SEQ ID No. 10 bis 13. Besonders bevorzugt weist die Prolin-4-hydroxylase die Aminosäuresequenz gemäß SEQ ID No. 10 auf, bevorzugt besteht aus dieser. Bevorzugt weist die Prolin-4-hydroxylase die Aminosäuresequenz gemäß SEQ ID No. 11 auf, bevorzugt besteht aus dieser. Bevorzugt weist die Prolin-4-hydroxylase die Aminosäuresequenz gemäß SEQ ID No. 12 auf, bevorzugt besteht aus dieser. Bevorzugt weist die Prolin-4-hydroxylase die Aminosäuresequenz gemäß SEQ ID No. 13 auf, bevorzugt besteht aus dieser. Proline 4-hydroxylase of bacterial origin is particularly preferred, in particular proline 4-hydroxylase is a proline 4-hydroxylase from Streptomyces griseoviridis, Dactylosporangium sp., Pseudomonas stutzeri, Bordetella bronchiseptica, Bradyrhizobium japonicum, Aeromonas cobaviae, Janthin sp. or Achromobacter xylosoxidans. In a particularly preferred embodiment, the proline-4-hydroxylase is a monomeric proline-4-hydroxylase. In a further preferred embodiment, the proline 4-hydroxylase has an amino acid sequence selected from SEQ ID no. 10 to 13, preferably consists of an amino acid sequence selected from SEQ ID NO. 10 to 13. The proline 4-hydroxylase particularly preferably has the amino acid sequence according to SEQ ID no. 10, preferably consists of this. The proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 11, preferably consists of this. The proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 12, preferably consists of this. The proline 4-hydroxylase preferably has the amino acid sequence according to SEQ ID no. 13, preferably consists of this.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung kann vorgesehen sein, dass in Schritt b) im Anschluss an die Transkription und Translation, also post-translational, die Hydrophobizität und/oder der Prolingehalt der exprimierten Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder Kollagenfusionspeptids, durch post-translationale
Modifizierung, insbesondere Hydroxylierungen, insbesondere Prolinhydroxylierungen und/oder Glykosylierungen, gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid verringert wird. In a further preferred embodiment of the present invention it can be provided that in step b) following the transcription and translation, i.e. post-translational, the hydrophobicity and / or the prolin content of the expressed collagen peptide component, in particular the collagen peptide or collagen fusion peptide, is carried out post-translational Modification, in particular hydroxylations, in particular proline hydroxylations and / or glycosylations, compared to which at least one collagen peptide encoded by the collagen peptide component is reduced.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung umfasst das in Schritt a) bereitgestellte prokaryotische Expressionssystem zusätzlich mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz und wird in Schritt b) in einem Kulturmedium unter Bedingungen kultiviert, die die Expression der mindestens einen rekombinanten Kollagenpeptid-Komponente und der mindestens einen Prolyl-4-hydroxylase ermöglichen, wobei die Kollagenpeptid-Komponente gegenüber dem mindestens einen von der Kollagenpeptid- Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist. In a further preferred embodiment of the present invention, the prokaryotic expression system provided in step a) additionally comprises at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase and is cultivated in step b) in a culture medium under conditions that promote the expression of the at least one recombinant collagen peptide Component and the at least one prolyl 4-hydroxylase, the collagen peptide component having a reduced hydrophobicity and / or a reduced prolin content compared to the at least one nucleotide sequence encoded by the collagen peptide component.
Bevorzugt handelt es sich bei dem in Schritt a) bereitgestellten prokaryoti sehen Expressionssystem um E. coli. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung handelt es sich bei dem in Schritt a) bereitgestellten prokaryoti sehen Expressionssystem um Bacillus subtilis. The prokaryotic expression system provided in step a) is preferably E. coli. In a further preferred embodiment of the present invention, the prokaryotic expression system provided in step a) is Bacillus subtilis.
Bevorzugt handelt es sich bei der mindestens eine mindestens eine rekombinante Kollagenpeptid- Komponente kodierende Nukleotidsequenz des prokaryoti sehen Expressionssystems um eine codon-optimierte Nukleinsäure, insbesondere um mindestens eine an die bevorzugte Codon- Verwendung des prokaryoti sehen Expressionssystems angepasste mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz. The at least one nucleotide sequence of the prokaryotic expression system coding for at least one recombinant collagen peptide component is preferably a codon-optimized nucleic acid, in particular at least one nucleotide sequence coding for the preferred codon use of the prokaryotic expression system .
Das erfindungsgemäße Verfahren ermöglicht es in vorteilhafter Weise, ein generell für das prokaryotische Expressionssystem, insbesondere in E. coli , toxisches rekombinantes Kollagenpeptid mit hoher Effizienz und in hoher Reinheit herzustellen. The method according to the invention advantageously enables a recombinant collagen peptide that is generally toxic to the prokaryotic expression system, in particular in E. coli, to be produced with high efficiency and in high purity.
Die erfindungsgemäße Verfahrensweise ist insofern besonders vorteilhaft, als dass rekombinante Kollagenpeptide auch in einem industriellen oder großtechnischen Verfahrensweg hergestellt werden können. In besonders bevorzugter Ausführungsform sind die erfmdungsgemäß bereitgestellten rekombinanten Kollagenpeptid-Komponenten biologisch wirksam. Insbesondere zeigen die mit dem erfmdungsgemäßen Verfahren hergestellten Kollagenpeptid-Komponenten eine biologische Wirksamkeit auf die Biosynthese von Proteinen der extrazellulären Matrix,
bevorzugt auf die Biosynthese von Kollagen, Elastin und/oder Proteoglykanen. Besonders bevorzugt zeigen die mit dem erfmdungsgemäßen Verfahren hergestellten Kollagenpeptid- Komponenten eine biologische Wirksamkeit auf Chondrozyten, Fibroblasten und/oder Osteoblasten. The procedure according to the invention is particularly advantageous in that recombinant collagen peptides can also be produced in an industrial or large-scale process. In a particularly preferred embodiment, the recombinant collagen peptide components provided according to the invention are biologically active. In particular, the collagen peptide components produced with the method according to the invention show a biological effectiveness on the biosynthesis of proteins of the extracellular matrix, preferably on the biosynthesis of collagen, elastin and / or proteoglycans. The collagen peptide components produced with the method according to the invention particularly preferably show a biological activity on chondrocytes, fibroblasts and / or osteoblasts.
Besonders bevorzugt ist die mindestens eine mindestens eine rekombinante Kollagenpeptid- Komponente kodierende Nukleotidsequenz codon-optimiert. The at least one nucleotide sequence coding for at least one recombinant collagen peptide component is particularly preferably codon-optimized.
Bevorzugt weist das mindestens eine durch die mindestens eine Nukleotidsequenz kodierte Kollagenpeptid eine in Kollagen der Typen I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, bevorzugt Typ I, II oder III, bevorzugt Typ I, bevorzugt Typ II, bevorzugt Typ III, vorkommende Aminosäuresequenz auf. The at least one collagen peptide encoded by the at least one nucleotide sequence preferably has a collagen of types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII , XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, preferably type I, II or III, preferably type I, preferably type II, preferably type III.
Bevorzugt weist das mindestens eine durch die mindestens eine Nukleotidsequenz kodierte Kollagenpeptid eine in Kollagen aus Wirbeltieren, insbesondere Fischen, Amphibien, Reptilien, Vögeln und Säugetieren, insbesondere in humanem, bovinem, porcinem, equinem oder avianem Kollagen der Typen I, II oder III, bevorzugt Typ I, bevorzugt Typ II, bevorzugt Typ III, vorkommende Aminosäuresequenz auf. The at least one collagen peptide encoded by the at least one nucleotide sequence preferably has one in collagen from vertebrates, in particular fish, amphibians, reptiles, birds and mammals, in particular in human, bovine, porcine, equine or avian collagen of types I, II or III Type I, preferably type II, preferably type III, occurring amino acid sequence.
Besonders bevorzugt weist das durch die Nukleotidsequenz kodierte Kollagenpeptid eine in bovinem Kollagen, insbesondere in bovinem Typ I Kollagen, bevorzugt in der al -Kette des bovinen Typ I Kollagen, vorkommende Aminosäuresequenz auf. The collagen peptide encoded by the nucleotide sequence particularly preferably has an amino acid sequence occurring in bovine collagen, in particular in bovine type I collagen, preferably in the a1 chain of bovine type I collagen.
Bevorzugt weist das Kollagenpeptid, insbesondere das Kollagenpeptid der Kollagenpeptid- Komponente oder das Kollagenpeptid des Kollagenfusionspeptids, mindestens eine Aminosäuresequenz ausgewählt aus der Gruppe bestehend aus SEQ ID No. 25, 26, 27, 28, 29, 30, 31 und 33, auf, bevorzugt besteht aus mindestens einer dieser. The collagen peptide, in particular the collagen peptide of the collagen peptide component or the collagen peptide of the collagen fusion peptide, preferably has at least one amino acid sequence selected from the group consisting of SEQ ID NO. 25, 26, 27, 28, 29, 30, 31 and 33, preferably consists of at least one of these.
Bevorzugt ist das durch die mindestens eine Nukleotidsequenz kodierte Kollagenpeptid des Kollagenfusionspeptids ein natürlich vorkommendes Kollagenpeptid. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist das durch die Nukleotidsequenz kodierte Kollagenpeptid des Kollagenfusionspeptids kein natürlich vorkommendes Kollagenpeptid. Bevorzugt ist das durch die Nukleotidsequenz kodierte Kollagenpeptid des Kollagenfusionspeptids ein gentechnisch verändertes Kollagenpeptid. In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung ist das durch die Nukleotidsequenz
kodierte Kollagenpeptid des Kollagenfusionspeptids ein gentechnisch verändertes Kollagenpeptid, bei dem mindestens eine Aminosäure der Aminosäuresequenz eines natürlich vorkommenden Kollagenpeptids, bevorzugt mindestens eine nicht-essentielle Aminosäure, insbesondere Ala, Asn, Asp, Glu, Ser, der Aminosäuresequenz eines natürlich vorkommenden Kollagenpeptids, durch mindestens eine ganz bestimmte Aminosäure, insbesondere durch mindestens eine essentielle Aminosäure, insbesondere Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, besonders bevorzugt Trp, ersetzt wurde. The collagen peptide of the collagen fusion peptide encoded by the at least one nucleotide sequence is preferably a naturally occurring collagen peptide. In a further preferred embodiment of the present invention, the collagen peptide of the collagen fusion peptide encoded by the nucleotide sequence is not a naturally occurring collagen peptide. The collagen peptide of the collagen fusion peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide. In a particularly preferred embodiment of the present invention, this is through the nucleotide sequence Coded collagen peptide of the collagen fusion peptide is a genetically modified collagen peptide in which at least one amino acid of the amino acid sequence of a naturally occurring collagen peptide, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser, the amino acid sequence of a naturally occurring collagen peptide, is replaced by at least one very specific amino acid, in particular by at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, has been replaced.
In einer bevorzugten Ausführungsform der vorliegenden Erfindung weist die rekombinante Kollagenpeptid-Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid-Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, eine Größe von 0,18 bis 110 kDa, bevorzugt 0,18 bis 100 kDa, bevorzugt 0,18 bis 90 kDa, bevorzugt 0,18 bis 80 kDa, bevorzugt 0,18 bis 70 kDa, bevorzugt 0,2 bis 60 kDa, bevorzugt 0,3 bis 50 kDa, bevorzugt 0,5 bis 50 kDa, bevorzugt 0,6 bis 50 kDa, bevorzugt 0,7 bis 50 kDa, bevorzugt 0,8 bis 50 kDa, bevorzugt 0,9 bis 50 kDa, bevorzugt 1 bis 50 kDa, bevorzugt 2 bis 50 kDa, bevorzugt 5 bis 50 kDa, bevorzugt 5 bis 40 kDa, bevorzugt 5 bis 30 kDa, bevorzugt 5 bis 20 kDa, bevorzugt 5 bis 10 kDa, auf. In a preferred embodiment of the present invention, the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a size of 0.18 to 110 kDa, preferably 0.18 to 100 kDa, preferably 0.18 to 90 kDa, preferably 0.18 to 80 kDa, preferably 0.18 to 70 kDa, preferably 0.2 to 60 kDa, preferably 0.3 to 50 kDa , preferably 0.5 to 50 kDa, preferably 0.6 to 50 kDa, preferably 0.7 to 50 kDa, preferably 0.8 to 50 kDa, preferably 0.9 to 50 kDa, preferably 1 to 50 kDa, preferably 2 to 50 kDa, preferably 5 to 50 kDa, preferably 5 to 40 kDa, preferably 5 to 30 kDa, preferably 5 to 20 kDa, preferably 5 to 10 kDa.
In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung weist die rekombinante Kollagenpeptid-Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid-Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, eine Größe von 0,18 bis 20 kDa, bevorzugt 0,2 bis 18 kDa, bevorzugt 0,3 bis 16 kDa, bevorzugt 0,5 bis 14 kDa, bevorzugt 0,6 bis 12 kDa, bevorzugt 0,8 bis 10 kDa, bevorzugt 1 bis 8 kDa, bevorzugt 1 bis 6 kDa, bevorzugt 1 bis 4 kDa, auf. In a particularly preferred embodiment of the present invention, the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a size of 0.18 up to 20 kDa, preferably 0.2 to 18 kDa, preferably 0.3 to 16 kDa, preferably 0.5 to 14 kDa, preferably 0.6 to 12 kDa, preferably 0.8 to 10 kDa, preferably 1 to 8 kDa, preferably 1 to 6 kDa, preferably 1 to 4 kDa.
In einer weiteren bevorzugten Ausführungsform weist die rekombinante Kollagenpeptid- Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid- Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, eine Größe von 10 bis 80 kDa, bevorzugt 10 bis 70 kDa, bevorzugt 10 bis 60 kDa, bevorzugt 10 bis 50 kDa, bevorzugt 10 bis 40 kDa, bevorzugt 10 bis 30 kDa, bevorzugt 10 bis 20 kDa, auf.
Besonders bevorzugt ist die rekombinante Kollagenpeptid-Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid-Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, hydroxyliert. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist die rekombinante Kollagenpeptid-Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid-Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, nicht hydroxyliert. In a further preferred embodiment, the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, preferably has a size of 10 to 80 kDa 10 to 70 kDa, preferably 10 to 60 kDa, preferably 10 to 50 kDa, preferably 10 to 40 kDa, preferably 10 to 30 kDa, preferably 10 to 20 kDa. The recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, is particularly preferably hydroxylated. In a further preferred embodiment of the present invention, the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, is not hydroxylated.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung weist die rekombinante Kollagenpeptid-Komponente, insbesondere die in Schritt c) gewonnene rekombinante Kollagenpeptid-Komponente, das Kollagenfusionspeptid oder das Kollagenpeptid, insbesondere das nach Abspaltung des mindestens einen Peptidrests erhaltene Kollagenpeptid, ein Verhältnis von Prolin zu Hydroxyprolin von 0% bis 45% Prolin zu 55% bis 100% Hydroxyprolin (bezogen auf Anzahl der Prolin- und Hydroxyprolinreste der Kollagenpeptid-Komponente) auf. In a further preferred embodiment of the present invention, the recombinant collagen peptide component, in particular the recombinant collagen peptide component obtained in step c), the collagen fusion peptide or the collagen peptide, in particular the collagen peptide obtained after cleavage of the at least one peptide residue, has a ratio of proline to hydroxyproline from 0% to 45% proline to 55% to 100% hydroxyproline (based on the number of proline and hydroxyproline residues of the collagen peptide component).
Die vorliegende Erfindung umfasst auch eine Kollagenpeptid-Komponente, herstellbar, insbesondere hergestellt, durch das erfmdungsgemäße Verfahren zur Herstellung einer rekombinanten Kollagenpeptid-Komponente in prokaryoti sehen Systemen. The present invention also comprises a collagen peptide component which can be produced, in particular produced, by the method according to the invention for producing a recombinant collagen peptide component in prokaryotic systems.
Die vorliegende Erfindung betrifft ferner ein Verfahren zur Herstellung eines rekombinanten hydroxylierten Kollagenpeptids in prokaryoti sehen Systemen, umfassend die Schritte: aa) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens ein Kollagenpeptid kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, bb) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression des mindestens einen Kollagenpeptids und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens eines hydroxylierten Kollagenpeptids ermöglichen, cc) Gewinnen mindestens eines hydroxylierten Kollagenpeptids, wobei das mindestens eine Kollagenpeptid das Aminosäuresequenzmotiv (Gly-X-Y)n aufweist und mindestens 50% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen.
Das erfindungsgemäße Verfahren erlaubt somit vorteilhafterweise die Herstellung mindestens eines hydroxylierten Kollagenpeptids mit einem Kollagen-typi sehen Hydroxylierungsmuster in prokaryoti sehen Systemen. The present invention also relates to a method for producing a recombinant hydroxylated collagen peptide in prokaryotic systems, comprising the steps: aa) providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one collagen peptide and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase , bb) cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide, cc) obtaining at least one hydroxylated collagen peptide, the at least one Collagen peptide has the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position. The method according to the invention thus advantageously allows the production of at least one hydroxylated collagen peptide with a collagen-type hydroxylation pattern see in prokaryotic systems.
In einer bevorzugten Ausführungsform der vorliegenden Erfindung liegen mindestens 50%, bevorzugt mindestens 55%, bevorzugt mindestens 60%, bevorzugt mindestens 65%, bevorzugt mindestens 70%, bevorzugt mindestens 75%, bevorzugt mindestens 80%, bevorzugt mindestens 85%, bevorzugt mindestens 90%, bevorzugt mindestens 95%, bevorzugt mindestens 100%, der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vor. In a preferred embodiment of the present invention, at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90 %, preferably at least 95%, preferably at least 100%, of the hydroxylations in the at least one collagen peptide on a proline in the Y position.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung besitzt das mindestens eine in Schritt cc) gewonnene Kollagenpeptid einen Hydroxylierungsgrad von 5 bis 100%, bevorzugt 5 bis 90%, bevorzugt 5 bis 80%, bevorzugt 10 bis 70%, bevorzugt 15 bis 60%, bevorzugt 20 bis 50%, bevorzugt 30 bis 50%, bevorzugt 35 bis 50%, bevorzugt 40 bis 50% (jeweils bezogen auf die Gesamtzahl der Prolin- und Lysinreste des Kollagenpeptids). In a further preferred embodiment of the present invention, the at least one collagen peptide obtained in step cc) has a degree of hydroxylation of 5 to 100%, preferably 5 to 90%, preferably 5 to 80%, preferably 10 to 70%, preferably 15 to 60%, preferably 20 to 50%, preferably 30 to 50%, preferably 35 to 50%, preferably 40 to 50% (each based on the total number of proline and lysine residues of the collagen peptide).
Bevorzugt besitzt das mindestens eine in Schritt cc) gewonnene Kollagenpeptid einen Hydroxylierungsgrad von mindestens 5%, bevorzugt mindestens 10%, bevorzugt mindestens 15%, bevorzugt mindestens 20%, bevorzugt mindestens 25%, bevorzugt mindestens 30%, bevorzugt mindestens 35%, bevorzugt mindestens 40%, bevorzugt mindestens 45%, bevorzugt mindestens 50% (jeweils bezogen auf die Gesamtzahl der Prolin- und Lysinreste des Kollagenpeptids). Bevorzugt weist das mindestens eine in Schritt cc) gewonnene Kollagenpeptid einen Hydroxylierungsgrad von höchstens 80%, bevorzugt höchstens 75%, bevorzugt höchstens 70%, bevorzugt höchstens 65%, bevorzugt höchstens 60%, bevorzugt höchstens 55%, bevorzugt höchstens 50%, auf (jeweils bezogen auf die Gesamtzahl der Prolin- und Lysinreste des Kollagenpeptids). The at least one collagen peptide obtained in step cc) preferably has a degree of hydroxylation of at least 5%, preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50% (each based on the total number of proline and lysine residues of the collagen peptide). The at least one collagen peptide obtained in step cc) preferably has a degree of hydroxylation of at most 80%, preferably at most 75%, preferably at most 70%, preferably at most 65%, preferably at most 60%, preferably at most 55%, preferably at most 50% ( each based on the total number of proline and lysine residues of the collagen peptide).
In besonders bevorzugter Ausführungsform der vorliegenden Erfindung handelt es sich bei der mindestens ein Kollagenpeptid kodierende Nukleotidsequenz, um eine natürlich vorkommende Nukleotidsequenz. Bevorzugt handelt es sich bei der mindestens ein Kollagenpeptid kodierende Nukleotidsequenz, um eine Nukleotidsequenz aus Säugetieren. Bevorzugt handelt es sich bei der mindestens ein Kollagenpeptid kodierende Nukleotidsequenz, um eine Nukleotidsequenz bovinen Ursprungs. Bevorzugt kodiert die Nukleotidsequenz ein natürlich vorkommendes Kollagenpeptid. In a particularly preferred embodiment of the present invention, the nucleotide sequence coding for at least one collagen peptide is a naturally occurring nucleotide sequence. The nucleotide sequence coding for at least one collagen peptide is preferably a nucleotide sequence from mammals. The nucleotide sequence coding for at least one collagen peptide is preferably a nucleotide sequence of bovine origin. The nucleotide sequence preferably encodes a naturally occurring collagen peptide.
Gemäß einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung handelt es sich bei der mindestens ein Kollagenpeptid kodierende Nukleotidsequenz, um keine natürlich
vorkommende Nukleotidsequenz. Bevorzugt handelt es sich bei der mindestens ein Kollagenpeptid kodierende Nukleotidsequenz, um eine gentechnisch veränderte Nukleotidsequenz. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung kodiert die Nukleotidsequenz kein natürlich vorkommendes Kollagenpeptid. Bevorzugt ist das durch die Nukleotidsequenz kodierte Kollagenpeptid ein gentechnisch verändertes Kollagenpeptid. In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung ist das durch die Nukleotidsequenz kodierte Kollagenpeptid ein gentechnisch verändertes Kollagenpeptid, bei dem mindestens eine Aminosäure der Aminosäuresequenz eines natürlich vorkommenden Kollagenpeptids, bevorzugt mindestens eine nicht-essentielle Aminosäure, insbesondere Ala, Asn, Asp, Glu, Ser, der Aminosäuresequenz eines natürlich vorkommenden Kollagenpeptids, durch mindestens eine ganz bestimmte Aminosäure, insbesondere durch mindestens eine essentielle Aminosäure, insbesondere Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, besonders bevorzugt Trp, ersetzt wurde. According to a further preferred embodiment of the present invention, the nucleotide sequence coding for at least one collagen peptide is not a natural one occurring nucleotide sequence. The nucleotide sequence coding for at least one collagen peptide is preferably a genetically modified nucleotide sequence. In a further preferred embodiment of the present invention, the nucleotide sequence does not encode a naturally occurring collagen peptide. The collagen peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide. In a particularly preferred embodiment of the present invention, the collagen peptide encoded by the nucleotide sequence is a genetically modified collagen peptide in which at least one amino acid of the amino acid sequence of a naturally occurring collagen peptide, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser , the amino acid sequence of a naturally occurring collagen peptide, is replaced by at least one very specific amino acid, in particular by at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp became.
In einer bevorzugten Ausführungsform der vorliegenden Erfindung handelt es sich bei der mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, um eine Nukleotidsequenz bakteriellen oder pflanzlichen Ursprungs. Bevorzugt handelt es sich bei mindestens eine Prolyl-4- hydroxylase kodierende Nukleotidsequenz, um eine Nukleotidsequenz bakteriellen Ursprungs. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung handelts es sich bei der mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, um eine Nukleotidsequenz pflanzlichen Ursprungs, bevorzugt um eine Nukleotid Sequenz aus Arabidopsis thaliana. Bevorzugt ist die mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz pflanzlichen Ursprungs, bevorzugt aus Arabidopsis thaliana , codon-optimiert. In a preferred embodiment of the present invention, the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of bacterial or plant origin. At least one nucleotide sequence coding for prolyl-4-hydroxylase is preferably a nucleotide sequence of bacterial origin. In a further preferred embodiment of the present invention, the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of plant origin, preferably a nucleotide sequence from Arabidopsis thaliana. The at least one prolyl-4-hydroxylase-coding nucleotide sequence of plant origin, preferably from Arabidopsis thaliana, is preferably codon-optimized.
Besonders bevorzugt weist die mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, die Nukleotidsequenz gemäß SEQ ID No. 14 auf. Bevorzugt weist die mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, mindestens 80%, bevorzugt mindestens 85%, bevorzugt mindestens 90%, bevorzugt mindestens 91%, bevorzugt mindestens 92%, bevorzugt mindestens 93%, bevorzugt mindestens 94%, bevorzugt mindestens 95%, bevorzugt mindestens 96%, bevorzugt mindestens 97%, bevorzugt mindestens 98%, bevorzugt mindestens 99%, Sequenzidentität zu der Nukleotidsequenz gemäß SEQ ID No. 14 auf. The at least one nucleotide sequence coding for prolyl 4-hydroxylase, the nucleotide sequence according to SEQ ID no. 14 on. The nucleotide sequence encoding at least one prolyl 4-hydroxylase preferably has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95 %, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the nucleotide sequence according to SEQ ID no. 14 on.
Besonders bevorzugt kodiert die mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz mindestens eine Prolyl-4-hyroxylase aus Arabidopsis thaliana , insbesondere
mindestens eine Prolyl-4-hydroxylase umfassend eine Aminosäuresequenz gemäß SEQ ID No. 15. The nucleotide sequence encoding at least one prolyl 4-hydroxylase particularly preferably encodes at least one prolyl 4-hydroxylase from Arabidopsis thaliana, in particular at least one prolyl 4-hydroxylase comprising an amino acid sequence according to SEQ ID no. 15th
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist die Prolyl-4- hydroxylase, insbesondere die Prolyl-4-hydroxylase aus Arabidopsis thaliana , ein Fusionsprotein. In bevorzugter Ausführungsform ist die Prolyl-4-hydroxylase, insbesondere die Prolyl-4- hydroxylase aus Arabidopsis thaliana , N-terminal mit MBP fusioniert. Besonders bevorzugt, weist die mindestens eine Prolyl-4-hydroxylase, insbesondere die Prolyl-4-hydroxylase aus Arabidopsis thaliana , eine Aminosäuresequenz gemäß SEQ ID No. 16 auf, bevorzugt besteht aus dieser. In a further preferred embodiment of the present invention, the prolyl-4-hydroxylase, in particular the prolyl-4-hydroxylase from Arabidopsis thaliana, is a fusion protein. In a preferred embodiment, the prolyl 4-hydroxylase, in particular the prolyl 4-hydroxylase from Arabidopsis thaliana, is N-terminally fused with MBP. Particularly preferably, the at least one prolyl 4-hydroxylase, in particular the prolyl 4-hydroxylase from Arabidopsis thaliana, has an amino acid sequence according to SEQ ID no. 16, preferably consists of this.
Die vorliegende Erfindung betrifft zudem ein hydroxyliertes Kollagenpeptid herstellbar, insbesondere hergestellt, durch das erfindungsgemäße Verfahren zur Herstellung eines rekombinanten hydroxylierten Kollagenpeptids in prokaryoti sehen Systemen. The present invention also relates to a hydroxylated collagen peptide which can be produced, in particular produced, by the method according to the invention for producing a recombinant hydroxylated collagen peptide in prokaryotic systems.
Die vorliegende Erfindung umfasst ferner ein Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend das Verfahren zur Herstellung einer rekombinanten Kollagenpeptid-Komponente in prokaryoti sehen Systemen gemäß der vorliegenden Erfindung, wobei das in Schritt a) bereitgestellte prokaryoti sehe Expressionssystem zusätzlich mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz umfasst und das prokaryoti sehe Expressionssystem in Schritt b) in einem Kulturmedium unter Bedingungen erfolgt, die die Expression des mindestens einen Kollagenpeptids und der mindestens einen Prolyl-4-hydroxylase ermöglichen. The present invention further comprises a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems, comprising the method for producing a recombinant collagen peptide component in prokaryotic systems according to the present invention, the prokaryotic expression system provided in step a) additionally comprises at least one nucleotide sequence coding for at least one prolyl 4-hydroxylase and the prokaryotic expression system in step b) is carried out in a culture medium under conditions which enable the expression of the at least one collagen peptide and the at least one prolyl 4-hydroxylase.
Demnach umfasst die vorliegende Erfindung insbesondere auch ein Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend die Schritte: i) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, ii) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid-
Komponente und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, iii) Gewinnen der hydroxylierten Kollagenpeptid-Komponente. Accordingly, the present invention in particular also comprises a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: i) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one at least one Nucleotide sequence coding for prolyl-4-hydroxylase, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, ii) culturing the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide Component and the at least one prolyl-4-hydroxylase enable to obtain at least one hydroxylated collagen peptide component, the collagen peptide component having a reduced hydrophobicity and / or a reduced prolin content compared to the at least one collagen peptide encoded by the collagen peptide component-encoding nucleotide sequence, iii ) Recovering the hydroxylated collagen peptide component.
In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung weist die in Schritt iii) gewonnene Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, das Aminosäuresequenzmotiv (Gly-X-Y)n auf, wobei mindestens 50% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. In a particularly preferred embodiment of the present invention, the collagen peptide component obtained in step iii), in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n , with at least 50% of the hydroxylations in the at least one collagen peptide on a proline in Y position are present.
Die vorliegende Erfindung betrifft insbesondere ein Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend die Schritte: a) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, b) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, c) Gewinnen der hydroxylierten Kollagenpeptid-Komponente,
wobei das mindestens eine Kollagenpeptid das Aminosäuresequenzmotiv (Gly-X-Y)n aufweist und mindestens 55% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. The present invention relates in particular to a method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: a) Providing a prokaryotic see expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one at least one prolyl 4-hydroxylase-coding nucleotide sequence, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, b) cultivating the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component and the at least one Enable prolyl 4-hydroxylase to obtain at least one hydroxylated collagen peptide component, the collagen peptide component opposite the at least one of the collagen peptide -Component-encoding nucleotide sequence-encoded collagen peptide has a reduced hydrophobicity and / or a reduced prolin content, c) recovery of the hydroxylated collagen peptide component, wherein the at least one collagen peptide has the amino acid sequence motif (Gly-XY) n and at least 55% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
Demnach handelt es sich bei der von der mindestens einen Nukleotidsequenz kodierten mindestens einen Prolyl-4-hydroxylase um eine Prolyl-4-Hydroxylase, die eine Spezifität, insbesondere vorwiegende Spezifität, für die Hydroxylierung von in Y-Position des Aminosäuresequenzmotivs (Gly-X-Y)n liegende Prolinreste aufweist. Durch die bevorzugte, insbesondere vorwiegende, Hydroxylierung von Prolinresten der Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder Kollagenfusionspeptids, in Y-Position des Aminosäuresequenzmotivs (Gly- X-Y)n wird vorteilhafterweise ein Hydroxylierungsmuster erzielt, das dem Hydroxylierungsmuster von Kollagen und Kollagenpeptiden von Wirbeltieren, insbesondere Fischen, Amphibien, Reptilien, Vögeln und Säugetieren, insbesondere von humanem, bovinem, porcinem, equinem oder avianem Kollagen und Kollagenpeptiden, bevorzugt von bovinem Kollagen und Kollagenpeptiden, ähnelt. Accordingly, the at least one prolyl-4-hydroxylase encoded by the at least one nucleotide sequence is a prolyl-4-hydroxylase which has a specificity, in particular predominant specificity, for the hydroxylation of in the Y position of the amino acid sequence motif (Gly-XY) Has n lying proline residues. The preferred, in particular predominant, hydroxylation of proline residues of the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, in the Y position of the amino acid sequence motif (Gly-XY) n advantageously achieves a hydroxylation pattern that corresponds to the hydroxylation pattern of collagen and collagen peptides from vertebrates, in particular Fish, amphibians, reptiles, birds and mammals, in particular human, bovine, porcine, equine or avian collagen and collagen peptides, preferably bovine collagen and collagen peptides.
In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung weist die Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, das Aminosäuresequenzmotiv (Gly-X-Y)n einmal, bevorzugt zweimal, bevorzugt dreimal, bevorzugt viermal, auf. Bevorzugt weist die Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, das Aminosäuresequenzmotiv (Gly-X-Y)n mindestens einmal, bevorzugt mindestens zweimal, bevorzugt mindestens dreimal, bevorzugt mindestens viermal, auf. In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung weist die Kollagenpeptid-Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, das Aminosäuresequenzmotiv (Gly-X-Y)n höchstens zweimal, bevorzugt höchstens dreimal, bevorzugt höchstens viermal, auf. In a particularly preferred embodiment of the present invention, the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n once, preferably twice, preferably three times, preferably four times. The collagen peptide component, in particular the collagen peptide or collagen fusion peptide , preferably has the amino acid sequence motif (Gly-XY) n at least once, preferably at least twice, preferably at least three times, preferably at least four times. In a further preferred embodiment of the present invention, the collagen peptide component, in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n at most twice, preferably at most three times, preferably at most four times.
Besonders bevorzugt ist n eine ganze Zahl > 1, bevorzugt > 2, bevorzugt > 3, bevorzugt > 4, bevorzugt > 5, bevorzugt > 6, bevorzugt > 7, bevorzugt > 8, bevorzugt > 9, bevorzugt > 10, bevorzugt > 15, bevorzugt > 20, bevorzugt > 25, bevorzugt > 30, bevorzugt > 35, bevorzugt > 40, bevorzugt > 45, bevorzugt > 50. N is particularly preferably an integer> 1, preferably> 2, preferably> 3, preferably> 4, preferably> 5, preferably> 6, preferably> 7, preferably> 8, preferably> 9, preferably> 10, preferably> 15, preferably> 20, preferably> 25, preferably> 30, preferably> 35, preferably> 40, preferably> 45, preferably> 50.
Erfindungsgemäß kann beispielsweise vorgesehen sein, dass das Aminosäuresequenzmotiv (Gly- X-Y)n in der Aminosäuresequenz der Kollagenpeptid-Komponente, insbesondere des
Kollagenpeptids oder Kollagenfusionspeptids, x-Mal, beispielsweise einmal, zweimal, dreimal oder viermal vorkommt, wobei n jeweils eine ganze Zahl > 1, bevorzugt > 2, bevorzugt > 3, bevorzugt > 4, bevorzugt > 5, bevorzugt > 6, bevorzugt > 7, bevorzugt > 8, bevorzugt > 9, bevorzugt > 10, bevorzugt > 15, bevorzugt > 20, bevorzugt > 25, bevorzugt > 30, bevorzugt > 35, bevorzugt > 40, bevorzugt > 45, bevorzugt > 50, ist. According to the invention it can be provided, for example, that the amino acid sequence motif (Gly-XY) n in the amino acid sequence of the collagen peptide component, in particular the Collagen peptide or collagen fusion peptide, occurs x times, for example once, twice, three times or four times, where n is an integer> 1, preferably> 2, preferably> 3, preferably> 4, preferably> 5, preferably> 6, preferably> 7 , preferably> 8, preferably> 9, preferably> 10, preferably> 15, preferably> 20, preferably> 25, preferably> 30, preferably> 35, preferably> 40, preferably> 45, preferably> 50.
Die vorliegende Erfindung betrifft auch eine hydroxylierte Kollagenpeptid-Komponente, herstellbar, insbesondere hergestellt, durch eines der erfmdungsgemäßen Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen. The present invention also relates to a hydroxylated collagen peptide component which can be produced, in particular produced, by one of the methods according to the invention for producing a recombinant hydroxylated collagen peptide component in prokaryotic systems.
Die vorstehend im Zusammenhang mit dem erfmdungsgemäßen Verfahren zur Herstellung einer rekombinanten Kollagenpeptid-Komponente in prokaryoti sehen Systemen getroffenen Aussagen und Ausführungsformen gelten mutatis mutandis auch für das Verfahren zur Herstellung eines rekombinanten hydroxylierten Kollagenpeptids in prokaryoti sehen Systemen und die Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Komponente in prokaryoti sehen Systemen und umgekehrt. The statements and embodiments made above in connection with the process according to the invention for producing a recombinant collagen peptide component in prokaryotic systems also apply mutatis mutandis to the process for producing a recombinant hydroxylated collagen peptide in prokaryotic systems and the process for producing a recombinant hydroxylated collagen peptide Component in prokaryoti see systems and vice versa.
Im Zusammenhang mit der vorliegenden Erfindung wird der Begriff „Kollagen“ in fachüblicher Weise verstanden, insbesondere so wie beispielsweise in der WO 01/34646 definiert. In bevorzugter Ausführungsform betrifft der Begriff „Kollagen“ die Kollagen-Typen I bis XXVII. In weiterer bevorzugter Ausführungsform wird unter dem Begriff „Kollagen“ ein die Sequenz Glycin-Prolin, Glycin-4-Hydroxyprolin oder Glycin-X-4-Hydroxyprolin, bevorzugt das repetitive Motiv (Gly-X-Y)n, aufweisendes Peptid verstanden, wobei X und Y jede Aminosäure sein können, vorzugsweise Prolin und 4-Hydroxylprolin sind. Besonders bevorzugt wird unter dem Begriff „Kollagen“ ein das repetitive Motiv (Gly-Pro-Y)n und/oder (Gly-X-Hyp)m aufweisendes Peptid verstanden, wobei X und Y jede Aminosäure sein können. In connection with the present invention, the term “collagen” is understood in a manner customary in the art, in particular as defined, for example, in WO 01/34646. In a preferred embodiment, the term “collagen” relates to collagen types I to XXVII. In a further preferred embodiment, the term “collagen” is understood to mean a peptide having the sequence glycine-proline, glycine-4-hydroxyproline or glycine-X-4-hydroxyproline, preferably the repetitive motif (Gly-XY) n, where X and Y can be any amino acid, preferably proline and 4-hydroxylproline. The term “collagen” is particularly preferably understood to mean a peptide having the repetitive motif (Gly-Pro-Y) n and / or (Gly-X-Hyp) m , where X and Y can be any amino acid.
Im Zusammenhang mit der vorliegenden Erfindung wird unter dem Begriff „Kollagenpeptid“ ein Protein oder Peptid verstanden, das eine in Kollagen gemäß vorstehender Definition vorkommende Aminosäuresequenz aufweist, wobei es sich bei dem Protein oder Peptid mindestens um ein Dipeptid, bevorzugt um ein Oligopeptid oder Polypeptid, handelt. Dabei kann das Kollagenpeptid insbesondere in chemisch-modifizierter Form, insbesondere hydroxylierter und/oder glykosylierter Form, vorliegen oder nicht modifiziert sein.
Ein „Kollagenpeptid“ im Sinne der vorliegenden Erfindung kann auch ein Kollagenprotein sein. Insbesondere kann das Kollagenpeptid der vorliegenden Erfindung in einzelsträngiger Form vorliegen, das Kollagenpeptid der vorliegenden Erfindung kann allerdings auch als Di- oder Trimer, insbesondere Trimer, aus gleichen oder verschiedenen Kollagenpeptiden vorliegen, insbesondere auch als tripelhelikales Kollagenpeptid. In connection with the present invention, the term “collagen peptide” is understood to mean a protein or peptide which has an amino acid sequence occurring in collagen according to the above definition, the protein or peptide being at least one dipeptide, preferably an oligopeptide or polypeptide, acts. The collagen peptide can in particular be present in chemically modified form, in particular hydroxylated and / or glycosylated form, or it can be unmodified. A “collagen peptide” within the meaning of the present invention can also be a collagen protein. In particular, the collagen peptide of the present invention can be present in single-stranded form, but the collagen peptide of the present invention can also be present as a dimer or trimer, in particular a trimer, from the same or different collagen peptides, in particular also as a triple-helical collagen peptide.
Im Zusammenhang mit der vorliegenden Erfindung wird unter einem „natürlich vorkommenden Kollagenpeptid“ ein unmittelbar aus natürlichen Quellen isolierbares Kollagenpeptid verstanden, also ein solches, das eine Aminosäuresequenz aufweist, wie sie in natürlich vorkommenden Nukleotidsequenzen eines Organismus kodiert werden, insbesondere ohne dass in diesen Nukleotidsequenzen Mutationen Vorkommen, insbesondere solche, die zu einem oder mehreren Aminosäure-Austäuschen führt. Insbesondere wird unter natürlich vorkommenden Kollagenpeptiden verstanden, dass diese natürlicherweise in einem Wirbeltier, insbesondere im Rind, oder einem Wirbellosen, insbesondere einer Qualle, Vorkommen. In besonders bevorzugter Ausführungsform ist ein natürlich vorkommendes Kollagenpeptid ein Kollagenpeptid, welches im Rind vorkommt. In connection with the present invention, a “naturally occurring collagen peptide” is understood to mean a collagen peptide that can be isolated directly from natural sources, that is, one that has an amino acid sequence as encoded in naturally occurring nucleotide sequences of an organism, in particular without mutations in these nucleotide sequences Occurrence, especially those that lead to one or more amino acid exchanges. In particular, naturally occurring collagen peptides are understood to mean that they occur naturally in a vertebrate, in particular in cattle, or in an invertebrate, in particular a jellyfish. In a particularly preferred embodiment, a naturally occurring collagen peptide is a collagen peptide which occurs in cattle.
Der Begriff „Kollagenpeptid-Komponente“ bezeichnet erfmdungsgemäß ein mindestens die Aminosäuresequenz eines Kollagenpeptids umfassendes Peptid. Dabei kann die Kollagenpeptid- Komponente in einer bevorzugten Ausführungsform aus der Aminosäuresequenz des Kollagenpeptids bestehen, also ein Kollagenpeptid sein. Es ist in einer weiteren Ausführungsform jedoch auch denkbar, dass die Kollagenpeptid-Komponente die Aminosäuresequenz mindestens eines Kollagenpeptids und mindestens eine weitere Aminosäuresequenz umfasst, bei der es sich nicht um ein Kollagenpeptid handelt. Gemäß dieser bevorzugten Ausführungsform handelt es sich bei der Kollagenpeptid-Komponente um ein Kollagenfusionspeptid. Bevorzugt weist die Kollagenpeptid-Komponente, insbesondere das Kollagenfusionspeptid, mindestens einen N- und/oder C-terminal an die Aminosäuresequenz des Kollagenpeptids fusionierten Peptidrest, insbesondere hydrophilen Peptidrest, auf. According to the invention, the term “collagen peptide component” denotes a peptide comprising at least the amino acid sequence of a collagen peptide. In a preferred embodiment, the collagen peptide component can consist of the amino acid sequence of the collagen peptide, that is to say it can be a collagen peptide. In a further embodiment, however, it is also conceivable that the collagen peptide component comprises the amino acid sequence of at least one collagen peptide and at least one further amino acid sequence that is not a collagen peptide. According to this preferred embodiment, the collagen peptide component is a collagen fusion peptide. The collagen peptide component, in particular the collagen fusion peptide, preferably has at least one peptide residue, in particular a hydrophilic peptide residue, fused N- and / or C-terminally to the amino acid sequence of the collagen peptide.
Unter einer „Wirtszelle“ wird erfmdungsgemäß eine lebende Zelle verstanden, die zur Expression von in Fremd-DNA, insbesondere in rekombinanter DNA, kodierten Peptiden oder Proteinen, befähigt ist. According to the invention, a “host cell” is understood to mean a living cell which is capable of expressing peptides or proteins encoded in foreign DNA, in particular in recombinant DNA.
Im Zusammenhang mit der vorliegenden Erfindung wird unter einer „rekombinanten Kollagenpeptid“ ein durch biotechnologische rekombinante Herstellung mittels eines
Expressionssystems gewonnenes Kollagenpeptid verstanden. Erfmdungsgemäß ist dem „rekombinanten Kollagenpeptid“ eigen, dass diese nicht aus natürlichen Quellen gewonnen werden. In connection with the present invention, a “recombinant collagen peptide” is a biotechnological recombinant production by means of a Expression system obtained collagen peptide understood. According to the invention, the “recombinant collagen peptide” is characteristic of not being obtained from natural sources.
Im Zusammenhang mit der vorliegenden Erfindung wird unter einer „Nukleotidsequenz“ die Abfolge der Nukleotide einer Nukleinsäure, insbesondere eines Polynukleinsäurestrangs, insbesondere eines DNA-Strangs, verstanden. Eine „Nukleotidsequenz“ ist daher sowohl als informatorische Einheit als auch als der diese Information physisch manifestierende DNA-Strang zu verstehen. In connection with the present invention, a “nucleotide sequence” is understood to mean the sequence of the nucleotides of a nucleic acid, in particular a polynucleic acid strand, in particular a DNA strand. A “nucleotide sequence” is therefore to be understood both as an informational unit and as the DNA strand that physically manifests this information.
Unter den Begriffen „Codon-Optimierung“ und „codon-optimiert“ wird im Zusammenhang mit der vorliegenden Erfindung die Anpassung der Nukleotidsequenz, insbesondere der für eine Aminosäure kodierenden Basentriplets, an die von dem gewählten Expressionssystem, insbesondere dem gewählten prokaryoti sehen Expressionssystem, bevorzugt verwendeten für eine bestimmte Aminosäure kodierenden Basentriplets, verstanden. Die „Codon-Optimierung“ basiert darauf, dass die verschiedenen für eine bestimmte Aminosäure kodierenden Basentriplets von unterschiedlichen Spezies bei der Proteinbiosynthese unterschiedlich häufig verwendet werden. Demnach führt eine „Codon-Optimierung“ zu einer Änderung der eine bestimmte Aminosäuresequenz kodierenden Nukleinsäure jedoch nicht zur Veränderung der kodierten Aminosäuresequenz selbst. In connection with the present invention, the terms “codon optimization” and “codon optimized” are preferably used to adapt the nucleotide sequence, in particular the base triplets coding for an amino acid, to the expression system selected by the selected expression system, in particular the selected prokaryotic expression system Base triplets coding for a specific amino acid understood. The “codon optimization” is based on the fact that the different base triplets coding for a specific amino acid are used differently by different species in protein biosynthesis. Accordingly, a “codon optimization” leads to a change in the nucleic acid coding for a specific amino acid sequence, but not to a change in the coded amino acid sequence itself.
Unter einer „Verringerung der Hydrophobizität“ wird erfmdungsgemäß eine Reduktion der Gesamt-Hydrophobizität der betreffenden Kollagenpeptid-Komponente im Vergleich zur Gesamt- Hydrophobizität des Kollagenpeptids der Kollagenpeptid-Komponente verstanden. Die „Verringerung der Hydrophobiziät“ kann erfmdungsgemäß beispielsweise durch Fusion des Kollagenpeptids mit mindestens einem Peptidrest, bevorzugt mit mindestens einem hydrophilen Peptidrest, erzielt werden. Ferner kann die Hydrophobizität der Kollagenpeptid-Komponente beispielsweise durch prä-translationale oder post-translationale Hydroxylierung von Prolin zu Hydroxyprolin verringert werden. Die Hydrophobizität des Peptids kann beispielsweise anhand des Eisenberg Consensus Scale (ECS), des Gunnar von Heijne Scale, des Kyte and Doolittle Scale, des Wimley-White Scale, des aWW Scale, des Engelman-Steitz Scale oder anderen dem Fachmann bekannten Bestimmungsverfahren ermittelt werden. Bevorzugt beträgt die Reduktion der Gesamt-Hydrophobizität der betreffenden Kollagenpeptid-Komponente im Vergleich zur Gesamt-Hydrophobizität des Kollagenpeptids der Kollagenpeptid-Komponente mindestens 5%, bevorzugt mindestens 10%, bevorzugt mindestens 20%, bevorzugt mindestens 30%, bevorzugt
mindestens 40%, bevorzugt mindestens 50%, bevorzugt mindestens 60%, bevorzugt mindestens 70%, bevorzugt mindestens 80%. According to the invention, a “reduction in the hydrophobicity” is understood to mean a reduction in the total hydrophobicity of the relevant collagen peptide component compared to the total hydrophobicity of the collagen peptide of the collagen peptide component. According to the invention, the “reduction in hydrophobicity” can be achieved, for example, by fusing the collagen peptide with at least one peptide residue, preferably with at least one hydrophilic peptide residue. Furthermore, the hydrophobicity of the collagen peptide component can be reduced, for example, by pre-translational or post-translational hydroxylation of proline to hydroxyproline. The hydrophobicity of the peptide can be determined, for example, using the Eisenberg Consensus Scale (ECS), the Gunnar von Heijne Scale, the Kyte and Doolittle Scale, the Wimley-White Scale, the aWW Scale, the Engelman-Steitz Scale or other determination methods known to the person skilled in the art . The reduction in the total hydrophobicity of the collagen peptide component in question compared to the total hydrophobicity of the collagen peptide of the collagen peptide component is preferably at least 5%, preferably at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%.
Die Bezeichnung „Verringerung des Prolingehalts“ bezeichnet erfindungsgemäß eine Reduktion des prozentualen Anteils der Prolinreste an der Gesamtzahl der Aminosäuren der betreffenden Kollagenpeptid-Komponente gegenüber dem prozentualen Anteil der Prolinreste an der Gesamtzahl der Aminosäuren in dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid. Eine „Verringerung des Prolingehalts“ der Kollagenpeptid-Komponente kann erfindungsgemäß beispielsweise dadurch erreicht werden, dass es sich bei der Kollagenpeptid-Komponente um ein Kollagenfusionspeptid handelt. Durch die Fusion mit mindestens einem Peptidrest wird der prozentuale Anteil an Prolinresten in der Kollagenpeptid-Komponente im Vergleich zum prozentualen Anteil in dem Prolin-reichen Kollagenpeptid gesenkt. Eine weitere Möglichkeit zur „Verringerung des Prolingehalts“ besteht in der prä- oder post-translationalen Hydroxylierung von Prolin zu Hydroxyprolin. Bevorzugt beträgt die Reduktion des prozentualen Anteils der Prolinreste an der Gesamtzahl der Aminosäuren der betreffenden Kollagenpeptid-Komponente gegenüber dem prozentualen Anteil der Prolinreste an der Gesamtzahl der Aminosäuren in dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid mindestens 5%, bevorzugt mindestens 10%, bevorzugt mindestens 20%, bevorzugt mindestens 30%, bevorzugt mindestens 40%, bevorzugt mindestens 50%, bevorzugt mindestens 60%, bevorzugt mindestens 70%, bevorzugt mindestens 80%. According to the invention, the term “reduction in the proline content” denotes a reduction in the percentage of proline residues in the total number of amino acids in the relevant collagen peptide component compared to the percentage of proline residues in relation to the total number of amino acids in the at least one collagen peptide encoded by the collagen peptide component . According to the invention, a “reduction in the prolin content” of the collagen peptide component can be achieved, for example, in that the collagen peptide component is a collagen fusion peptide. As a result of the fusion with at least one peptide residue, the percentage of proline residues in the collagen peptide component is reduced compared to the percentage in the proline-rich collagen peptide. Another possibility for “reducing the proline content” is the pre- or post-translational hydroxylation of proline to hydroxyproline. The reduction of the percentage of proline residues in the total number of amino acids of the relevant collagen peptide component compared to the percentage of proline residues in relation to the total number of amino acids in the at least one collagen peptide encoded by the collagen peptide component is preferably at least 5%, preferably at least 10 %, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%.
Der Begriff „Hydroxylierungsgrad“ bezeichnet erfindungsgemäß den prozentualen Anteil der hydroxylierten Prolin- und Lysinreste an der Gesamtzahl der Prolin- und Lysinreste der Kollagenpeptid-Komponente, insbesondere des Kollagenpeptids oder des Kollagenfusionspeptids. According to the invention, the term “degree of hydroxylation” denotes the percentage of hydroxylated proline and lysine residues in the total number of proline and lysine residues of the collagen peptide component, in particular of the collagen peptide or the collagen fusion peptide.
Im Zusammenhang mit der vorliegenden Erfindung wird unter einem „Expressionssystem“ ein System verstanden, in dem eine gezielte und kontrollierte Proteinbiosynthese erfolgen kann. Unter einem „prokaryoti sehen Expressionssystem“ ist erfindungsgemäße ein zellbasiertes biologische System zu verstehen, das in der Lage ist, gezielt und kontrolliert Proteinbiosynthese zu betreiben, das heißt ausgehend von einer Nukleinsäure als Informationsträger Peptide oder Proteine zu synthetisieren. In connection with the present invention, an “expression system” is understood to mean a system in which a targeted and controlled protein biosynthesis can take place. A “prokaryotic expression system” is to be understood according to the invention as a cell-based biological system which is able to carry out protein biosynthesis in a targeted and controlled manner, that is to say to synthesize peptides or proteins starting from a nucleic acid as an information carrier.
Die „Expression“ einer Nukleotidsequenz umfasst im Zusammenhang mit der vorliegenden Erfindung insbesondere die Transkription in mRNA (messenger-RNA) und die anschließende
Translation in ein Peptid oder, im Zusammenhang mit der vorliegenden Erfindung gleichbedeutend, auch Protein. In connection with the present invention, the “expression” of a nucleotide sequence includes, in particular, the transcription into mRNA (messenger RNA) and the subsequent transcription Translation into a peptide or, synonymous in connection with the present invention, also protein.
Unter „Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente zum Erhalt mindestens einer Kollagenpeptid-Komponente ermöglichen“, „Bedingungen, die die Expression des mindestens einen Kollagenpeptids und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens eines hydroxylierten Kollagenpeptids ermöglichen“, „Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen“ und „Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid-Komponente und der mindestens einen Prolin-4-hydroxylase (PIN4H) zum Erhalt mindestens einer hydroxylierten Kollagenpeptid- Komponente ermöglichen“ werden erfmdungsgemäß Bedingungen, wie insbesondere Temperatur, Druck, Zeit, Licht sowie An- oder Abwesenheit von Induktoren und/oder Repressoren, verstanden, die eine Expression des betreffenden Peptids und/oder Proteins aktivieren oder verstärken. In einer bevorzugten Ausführungsform erfolgt die Expression des betreffenden Peptids und/oder Proteins in Rahmen einer Hochzelldichtefermentation, insbesondere unter Hochdruck, bevorzugt Luft- Hochdruck. Die konkreten Bedingungen, die eine Expression des betreffenden Peptids und/oder Proteins ermöglichen, sind dem Fachmann bekannt und hängen von dem verwendeten Expressionssystem und der verwendeten Expressionskassette, insbesondere des darin enthaltenen Promotors, ab. Bei der Expression des betreffenden Peptids und/oder Proteins kann es sich je nach Aufbau der Expressionskassette um eine konstitutive oder induzierbare Expression handeln. Under “conditions that enable the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component”, “conditions that enable the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide "," Conditions that enable the expression of the at least one recombinant collagen peptide component and the at least one prolyl 4-hydroxylase to obtain at least one hydroxylated collagen peptide component "and" Conditions that enable the expression of the at least one recombinant collagen peptide component and the enable at least one proline-4-hydroxylase (PIN4H) to obtain at least one hydroxylated collagen peptide component “according to the invention, conditions such as temperature, pressure, time, light and the presence or absence of inducers and / or repressors, are understood to mean that expression of the b Activate or amplify the relevant peptide and / or protein. In a preferred embodiment, the peptide and / or protein in question is expressed in the context of a high cell density fermentation, in particular under high pressure, preferably high pressure air. The specific conditions which enable expression of the peptide and / or protein in question are known to the person skilled in the art and depend on the expression system used and the expression cassette used, in particular the promoter contained therein. The expression of the peptide and / or protein in question can be constitutive or inducible expression, depending on the structure of the expression cassette.
Im Zusammenhang mit der vorliegenden Erfindung wird unter „Gewinnen eines rekombinanten Kollagenfusionspeptids“, „Gewinnen eines rekombinanten Kollagenpeptids“, „Gewinnen eines hydroxylierten rekombinanten Kollagenpeptids“ oder „Gewinnen einer hydroxylierten Kollagenpeptid-Komponente“ die Isolierung einer Kollagenpeptid-Komponente, eines Kollagenpeptids oder eines Kollagenfusionspeptids aus einer mehrere Komponenten enthaltenden Zusammensetzung mittels bekannter Isolierungsverfahren, wie beispielsweise Zentrifugationsverfahren, insbesondere differenzielle Zentrifugation und/oder Dichtegradientenzentrifugation, chromatografischer Verfahren, insbesondere Geifiltrations-, Ionenaustausch-, Affinitäts- und/oder Hochleistungsflüssigkeitschromatografie, Elektrophoreseverfahren, Filtrationsverfahren und/oder Extraktionsverfahren, verstanden, wobei eine Anreicherung und Reinigung der betreffenden Komponente aus der mehrere Komponenten
enthaltenden Zusammensetzung bevorzugt durch sequenzielle Anwendung mehrerer Isolationsverfahren erzielt werden kann. In the context of the present invention, “obtaining a recombinant collagen fusion peptide”, “obtaining a recombinant collagen peptide”, “obtaining a hydroxylated recombinant collagen peptide” or “obtaining a hydroxylated collagen peptide component” refers to the isolation of a collagen peptide component, a collagen peptide or a collagen fusion peptide from a composition containing several components by means of known isolation processes, such as centrifugation processes, in particular differential centrifugation and / or density gradient centrifugation, chromatographic processes, in particular geifiltration, ion exchange, affinity and / or high-performance liquid chromatography, electrophoresis processes, filtration processes and / or extraction processes, where an enrichment and purification of the component in question from the several components containing composition can preferably be achieved by sequential application of several isolation methods.
Im Zusammenhang mit der vorliegenden Erfindung wird unter den Begriffen „umfassend“ und „aufweisend“ verstanden, dass zusätzlich zu den von diesen Begriffen explizit erfassten Elementen noch weitere, nicht explizit genannte Elemente hinzutreten können. Im Zusammenhang mit der vorliegenden Erfindung wird unter diesen Begriffen auch verstanden, dass allein die explizit genannten Elemente erfasst werden und keine weiteren Elemente vorliegen. In dieser besonderen Ausführungsform ist die Bedeutung der Begriffe „umfassend“ und „aufweisend“ gleichbedeutend mit dem Begriff „bestehend aus“. Darüber hinaus erfassen die Begriffe „umfassend“ und „aufweisend“ auch Zusammensetzungen, die neben den explizit genannten Elementen auch weitere nicht genannte Elemente enthalten, die jedoch von funktionell und qualitativ untergeordneter Natur sind. In dieser Ausführungsform sind die Begriffe „umfassend“ und „aufweisend“ gleichbedeutend mit dem Begriff „im Wesentlichen bestehend aus“. In connection with the present invention, the terms “comprising” and “having” are understood to mean that, in addition to the elements explicitly covered by these terms, further elements that are not explicitly mentioned can be added. In connection with the present invention, these terms are also understood to mean that only the explicitly mentioned elements are included and no further elements are present. In this particular embodiment, the meaning of the terms “comprising” and “having” is synonymous with the term “consisting of”. In addition, the terms “comprising” and “having” also include compositions which, in addition to the explicitly named elements, also contain other non-named elements, which are, however, of a functionally and qualitatively subordinate nature. In this embodiment, the terms “comprising” and “having” are synonymous with the term “consisting essentially of”.
Sofern im Zusammenhang mit der vorliegenden Erfindung die erste und zweite Nachkommastelle oder die zweite Nachkommastelle nicht angegeben sind/ist, sind/ist diese als 0 zu setzen. If, in connection with the present invention, the first and second decimal places or the second decimal places are / is not specified, these are / is to be set as 0.
Unter dem Begriff „und/oder“ wird in Zusammenhang mit der vorliegenden Erfindung verstanden, dass alle Mitglieder einer Gruppe, welche durch den Begriff „und/oder“ verbunden sind, sowohl alternativ zueinander als auch jeweils untereinander kumulativ in einer beliebigen Kombination offenbart sind. Dies bedeutet für den Ausdruck „A, B und/oder C“, dass folgender Offenbarungsgehalt darunter zu verstehen ist: a) A oder B oder C oder b) (A und B) oder c) (A und C) oder d) (B und C) oder e) (A und B und C). In connection with the present invention, the term “and / or” is understood to mean that all members of a group which are connected by the term “and / or” are disclosed both as alternatives to one another and also cumulatively with one another in any combination. For the expression "A, B and / or C" this means that the following disclosure content is to be understood: a) A or B or C or b) (A and B) or c) (A and C) or d) ( B and C) or e) (A and B and C).
Weitere bevorzugte Ausführungsformen der vorliegenden Erfindung ergeben sich aus den folgenden Aspekten und aus den Unteransprüchen. Further preferred embodiments of the present invention emerge from the following aspects and from the subclaims.
Aspekt 1: Verfahren zur Herstellung einer rekombinanten Kollagenpeptid-Komponente in prokaryoti sehen Systemen, umfassend die Schritte: a) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst,
b) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente zum Erhalt mindestens einer Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid-Komponente gegenüber dem mindestens einen von der Kollagenpeptid- Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, c) Gewinnen der Kollagenpeptid-Komponente. Aspect 1: A method for producing a recombinant collagen peptide component in prokaryotic systems, comprising the steps: a) Providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component, the nucleotide sequence coding for the at least one recombinant collagen peptide component the nucleotide sequence comprises at least one collagen peptide, b) Cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one recombinant collagen peptide component to obtain at least one collagen peptide component, the collagen peptide component being compared to the at least one collagen peptide encoded by the collagen peptide component has a reduced hydrophobicity and / or a reduced proline content, c) recovering the collagen peptide component.
Aspekt 2: Verfahren nach Aspekt 1, wobei in der die Kollagenpeptid-Komponente kodierenden Nukleotidsequenz des prokaryoti sehen Expressionssystems die ein Kollagenpeptid kodierende Nukleotidsequenz mit mindestens einer Nukleotidsequenz fusioniert ist, die einen Peptidrest kodiert. Aspect 2: The method according to aspect 1, wherein in the nucleotide sequence of the prokaryotic expression system that codes for the collagen peptide component, the nucleotide sequence that codes for a collagen peptide is fused with at least one nucleotide sequence that codes for a peptide residue.
Aspekt 3: Verfahren nach Aspekt 2, wobei der Peptidrest mindestens einen Protein-tag, einAspect 3: The method according to aspect 2, wherein the peptide residue is at least one protein tag
Signalpeptid und/oder eine Kaschierungsdomäne aufweist. Having signal peptide and / or a lamination domain.
Aspekt 4: Verfahren nach einem der Aspekte 2 und 3, wobei der Peptidrest abspaltbar ist, insbesondere enzymatisch abspaltbar. Aspect 4: Method according to one of aspects 2 and 3, wherein the peptide residue can be split off, in particular can be split off enzymatically.
Aspekt 5: Verfahren nach einem der Aspekte 2 bis 4, wobei nach Schritt c) oder nach Schritt b) und vor Schritt c) eine Abspaltung des mindestens einen Peptidrests der Kollagenpeptid- Komponente erfolgt. Aspect 5: Method according to one of aspects 2 to 4, wherein after step c) or after step b) and before step c) the at least one peptide residue of the collagen peptide component is split off.
Aspekt 6: Verfahren nach einem der Aspekte 2 bis 5, wobei die Kollagenpeptid-Komponente ein Kollagenpeptid und mindestens ein N- und/oder C-terminales Sekretions-Signalpeptid umfasst. Aspect 6: The method according to any one of aspects 2 to 5, wherein the collagen peptide component comprises a collagen peptide and at least one N- and / or C-terminal secretion signal peptide.
Aspekt 7: Verfahren nach einem der Aspekte 1 bis 6, wobei das durch die Nukleotidsequenz kodierte Kollagenpeptid eine in bovinem Kollagen, insbesondere in bovinem Typ I Kollagen, bevorzugt in der al -Kette des bovinen Typ I Kollagen, vorkommende Aminosäuresequenz aufweist.
Aspekt 8: Verfahren zur Herstellung eines rekombinanten hydroxylierten Kollagenpeptids in prokaryoti sehen Systemen, umfassend die Schritte: aa) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens ein Kollagenpeptid kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, bb) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression des mindestens einen Kollagenpeptids und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens eines hydroxylierten Kollagenpeptids ermöglichen, cc) Gewinnen mindestens eines hydroxylierten Kollagenpeptids, wobei das mindestens eine Kollagenpeptid das Aminosäuresequenzmotiv (Gly-X-Y)n aufweist und mindestens 50% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. Aspect 7: Method according to one of aspects 1 to 6, wherein the collagen peptide encoded by the nucleotide sequence has an amino acid sequence occurring in bovine collagen, in particular in bovine type I collagen, preferably in the a1 chain of bovine type I collagen. Aspect 8: A method for producing a recombinant hydroxylated collagen peptide in prokaryotic systems, comprising the steps: aa) providing a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one collagen peptide and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase, bb) Cultivating the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one collagen peptide and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide, cc) obtaining at least one hydroxylated collagen peptide, the at least one collagen peptide having the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
Aspekt 9: Verfahren nach Aspekt 8, wobei mindestens 80% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. Aspect 9: The method according to aspect 8, wherein at least 80% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
Aspekt 10: Verfahren nach Aspekt 8 oder 9, wobei das das mindestens eine in Schritt cc) gewonnene Kollagenpeptid einen Hydroxylierungsgrad von mindestens 5% aufweist (bezogen auf die Gesamtzahl der Prolin- und Lysinreste des Kollagenpeptids). Aspect 10: The method according to aspect 8 or 9, wherein the at least one collagen peptide obtained in step cc) has a degree of hydroxylation of at least 5% (based on the total number of proline and lysine residues of the collagen peptide).
Aspekt 11 : Verfahren nach einem der Aspekte 8 bis 10, wobei es sich bei der mindestens einen Prolyl-4-hydroxylase kodierende Nukleotidsequenz, um eine Nukleotidsequenz bakteriellen oder pflanzlichen Ursprungs handelt. Aspect 11: The method according to any one of aspects 8 to 10, wherein the at least one nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence of bacterial or plant origin.
Aspekt 12: Verfahren nach Aspekt 11, wobei es sich bei der Prolyl-4-hydroxylase kodierenden Nukleotidsequenz um eine Nukleotidsequenz aus Arabidopsis thaliana handelt. Aspect 12: The method according to aspect 11, wherein the nucleotide sequence coding for prolyl 4-hydroxylase is a nucleotide sequence from Arabidopsis thaliana.
Aspekt 13: Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid-Aspect 13: Method for producing a recombinant hydroxylated collagen peptide
Komponente in prokaryoti sehen Systemen, umfassend die Schritte:
i) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, ii) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, iii) Gewinnen der hydroxylierten Kollagenpeptid-Komponente. Component in prokaryoti see systems, comprising the steps: i) Provision of a prokaryotic expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one nucleotide sequence coding for at least one prolyl-4-hydroxylase, the nucleotide sequence coding for at least one recombinant collagen peptide component comprising the nucleotide sequence of at least one collagen peptide, ii ) Cultivating the prokaryoti expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component and the at least one prolyl-4-hydroxylase to obtain at least one hydroxylated collagen peptide component, the collagen peptide component compared to the at least one collagen peptide encoded by the collagen peptide component-encoding nucleotide sequence has a reduced hydrophobicity and / or a reduced prolin content, iii) recovery of the hydroxylated collagen p eptid component.
Aspekt 14: Verfahren nach Aspekt 13, wobei die in Schritt iii) gewonnene Kollagenpeptid- Komponente, insbesondere das Kollagenpeptid oder Kollagenfusionspeptid, das Aminosäuresequenzmotiv (Gly-X-Y)n aufweist und mindestens 50% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. Aspect 14: The method according to aspect 13, wherein the collagen peptide component obtained in step iii), in particular the collagen peptide or collagen fusion peptide, has the amino acid sequence motif (Gly-XY) n and at least 50% of the hydroxylations in the at least one collagen peptide on a proline in Y Position are available.
Aspekt 15: Kollagenpeptid-Komponente, hergestellt durch ein Verfahren gemäß einem der Aspekte 1 bis 7. Aspect 15: Collagen peptide component produced by a method according to any one of Aspects 1 to 7.
Aspekt 16: Hydroxyliertes Kollagenpeptid, hergestellt durch ein Verfahren gemäß einem der Aspekte 8 bis 12. Aspect 16: Hydroxylated collagen peptide produced by a method according to any one of Aspects 8 to 12.
Aspekt 17: Hydroxylierte Kollagenpeptid-Komponente, hergestellt durch das Verfahren gemäß einem der Aspekte 13 und 14. Aspect 17: Hydroxylated collagen peptide component produced by the method according to any one of Aspects 13 and 14.
Die Erfindung wird nachstehend ohne Beschränkung des allgemeinen Erfmdungsgedankens anhand von Ausführungsbeispielen illustriert. The invention is illustrated below with the aid of exemplary embodiments, without restricting the general inventive concept.
Beispiel 1:
Cvtosolische Expression einer Kollagenpeptid-Komponente in E. coli Example 1: Cytosolic expression of a collagen peptide component in E. coli
1. Transformation der Zellen 1. Transformation of the cells
Für jede Expression wird 1 pL des Vektor pMAL-c5x (50-100 ng/pL) umfassend ein Tetracyclin- Resistenzgen und eine der SEQ ID No. 19 und 20, zu 50 pL auf Eis aufgetauten, chemisch kompetenten E. coli BL21 gegeben und für 30 min auf Eis gelagert. Anschließend erfolgt ein Hitzeschock für 30 s bei 42 °C, woraufhin die Zellen direkt für 5 min auf Eis gekühlt werden, bevor 950 pL SOC-Medium (20 g/L Sojapepton, 5 g/L Hefeextrakt, 0,6 g/L NaCl, 0,2 g/L KCl, 10 mL/L 1 M MgCh, 10 mL/L 1 M MgSCk, 10 mL/L 2 M Glukose) zu den Zellen gegeben wird. Die Zellen werden in einem Überkopf Schüttler für ca. 70 min bei 37 °C kultiviert. Anschließend werden die Zellen durch Zentrifugation bei 7.000 g für 2 min bei 4 °C zentrifugiert. Der Überstand wird bis auf ca. 100 pL, in denen das Zellpellet resuspendiert wird, verworfen. Die Zellen werden anschließend auf einer bei 37 °C vorgewärmten LB Agarplatte mit 25 mg/L Tetracyclin ausgestrichen und über Nacht bei 37 °C bebrütet. For each expression, 1 pL of the vector pMAL-c5x (50-100 ng / pL) comprising a tetracycline resistance gene and one of SEQ ID no. 19 and 20, added to 50 pL thawed on ice, chemically competent E. coli BL21 and stored on ice for 30 min. This is followed by a heat shock for 30 s at 42 ° C., after which the cells are cooled directly on ice for 5 min before using 950 μl of SOC medium (20 g / L soy peptone, 5 g / L yeast extract, 0.6 g / L NaCl , 0.2 g / L KCl, 10 mL / L 1 M MgCh, 10 mL / L 1 M MgSCk, 10 mL / L 2 M glucose) is added to the cells. The cells are cultivated in an overhead shaker for approx. 70 min at 37 ° C. The cells are then centrifuged by centrifugation at 7,000 g for 2 min at 4 ° C. The supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended. The cells are then streaked with 25 mg / L tetracycline on an LB agar plate preheated to 37 ° C. and incubated at 37 ° C. overnight.
2. Kultivierung und Induktion der Expression 2. Cultivation and induction of expression
Am nächsten Tag wird eine einzelne Kolonie gepickt und zur Inokulation von 30 mL LB-Medium (10 g/L NaCl, 10 g/L Sojapepton, 5 g/L Hefeextrakt, pH 7,4) mit 25 mg/L Tetracyclin in einem 300 mL Schüttelkolben mit Schikanen genutzt. Die Kultur wird bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler über Nacht kultiviert. The next day, a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 25 mg / L tetracycline in a 300 mL shake flasks with baffles are used. The culture is cultivated overnight on an orbital shaker at 37 ° C. and 120 revolutions per minute (deflection 25 mm).
Am darauf folgenden Tag werden 200 mL TB-Medium (4 g/L Glycerin, 12 g/L Sojapepton, 24 g/L Hefeextrakt, 2,31 g/L KH2PO4, 12,54 g/L K2HPO4, pH 7,4) mit 25 mg/L Tetracyclin in einem 1 L Schüttelkolben mit Schikanen unter Verwendung der Vorkultur auf eine optische Dichte von OD600 = 0,1 angeimpft und bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler kultiviert. Für die Expression im Schüttelkolben wird bei einer optischen Dichte von OD600 = 2-3 mit 0,1 mM IPTG induziert und die Kultivierung bei 28 °C fortgesetzt, bevor nach 20-22 h die Kultivierung durch Zellemte abgebrochen wird. The following day, 200 mL TB medium (4 g / L glycerine, 12 g / L soy peptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, pH 7.4) inoculated with 25 mg / L tetracycline in a 1 L shake flask with baffles using the preculture to an optical density of OD600 = 0.1 and cultivated at 37 ° C and 120 revolutions per minute (deflection 25 mm) on an orbital shaker. For expression in the shake flask, induction is carried out at an optical density of OD600 = 2-3 with 0.1 mM IPTG and cultivation is continued at 28 ° C., before cultivation is terminated by cells after 20-22 h.
Erfolgte die Expression nicht im Schüttelkolben sondern erst im Fermenter, so wird diese zuvor angesetzte Kultur nicht induziert und über Nacht bei 37 °C kultiviert und als die Vorkultur 2. Stufe verwendet.
Am nächsten Tag wird ein Bioreaktor mit 12 L TB-Medium und 25 mg/L Tetracyclin mit der Vorkultur 2. Stufe auf eine OD600 von ca. 0,1 eingestellt. Die Rührerdrehfrequenz startet bei 200 Umdrehungen pro Minute und steigt entsprechend einer Rührkaskade bei Unterschreitung des relativen pCh-Werts von 60 % um jeweils 2 % bis die maximale Rührerdrehfrequenz von 1500 Umdrehungen pro Minute erreicht wird. Zur Begasung werden 12 Normliter Luft pro Minute verwendet und ein Druck von 0,2 bar eingestellt. Der pH-Wert wird mit 10 %-iger Phosphorsäure und 4 M Natriumhydroxid-Lösung konstant auf 7,0 gehalten. Die Kultivierung erfolgt bei 37 °C. Bei einer optischen Dichte von OD600 = 9,0-10,0 wird mit 0,1 mM IPTG induziert. Die Kultivierung wird nach Erreichen der stationären Phase beendet. If the expression did not take place in the shake flask but only in the fermenter, this previously set culture is not induced and cultivated overnight at 37 ° C. and used as the second stage preculture. The next day, a bioreactor with 12 L TB medium and 25 mg / L tetracycline with the second stage preculture is set to an OD600 of approx. 0.1. The stirrer rotation frequency starts at 200 revolutions per minute and increases according to a stirring cascade when the relative pCh value falls below 60% by 2% each time until the maximum stirrer rotation frequency of 1500 revolutions per minute is reached. For gassing, 12 standard liters of air per minute are used and a pressure of 0.2 bar is set. The pH is kept constant at 7.0 with 10% phosphoric acid and 4 M sodium hydroxide solution. Cultivation takes place at 37 ° C. At an optical density of OD600 = 9.0-10.0, 0.1 mM IPTG is induced. The cultivation is ended after the stationary phase has been reached.
Beispiel 2: Example 2:
Sekretorische Expression einer Kollagenpeptid-Komponente in E. coli mittels des SignalpeptidsSecretory expression of a collagen peptide component in E. coli using the signal peptide
HlvA HlvA
1. Transformation der Zellen 1. Transformation of the cells
Für jede Expression wird 1 pL des Vektors pBR322, umfassend ein Tetracyclin-Resistenzgen und eine der SEQ ID No. 21, für die untersuchten Kollagenpeptid-Komponenten (50-100 ng/pL) und 1 pL des pACYC_HlyB+D (50-100 ng/pL) zur bicistronischen Expression der Porenproteine zu 50 pL auf Eis aufgetauten, chemisch kompetenten E. coli BL21(DE3) gegeben und für 30 min auf Eis gelagert. Anschließend erfolgt ein Hitzeschock für 30 s bei 42 °C, woraufhin die Zellen direkt für 5 min auf Eis gekühlt werden, bevor 950 pL SOC-Medium (20 g/L Sojapepton, 5 g/L Hefeextrakt, 0,6 g/L NaCl, 0,2 g/L KCl, 10 mL/L 1 M MgCh, 10 mL/L 1 M MgS02, 10 mL/L 2 M Glukose) zu den Zellen gegeben wird. Die Zellen werden in einem Überkopfschüttler für ca. 70 min bei 37 °C kultiviert. Anschließend werden die Zellen durch Zentrifugation bei 7.000 g für 2 min bei 4 °C zentrifugiert. Der Überstand wird bis auf ca. 100 pL, in denen das Zellpellet resuspendiert wird, verworfen. Die Zellen werden anschließend auf einer bei 37 °C vorgewärmten LB Agarplatte mit 20 mg/L Tetracyclin und 20 mg/L Chloramphenicol ausgestrichen und über Nacht bei 37 °C bebrütet. For each expression, 1 pL of the vector pBR322, comprising a tetracycline resistance gene and one of SEQ ID no. 21, for the examined collagen peptide components (50-100 ng / pL) and 1 pL of pACYC_HlyB + D (50-100 ng / pL) for bicistronic expression of the pore proteins to 50 pL thawed on ice, chemically competent E. coli BL21 ( DE3) and stored on ice for 30 min. This is followed by a heat shock for 30 s at 42 ° C., after which the cells are cooled directly on ice for 5 min before using 950 μl of SOC medium (20 g / L soy peptone, 5 g / L yeast extract, 0.6 g / L NaCl , 0.2 g / L KCl, 10 mL / L 1 M MgCh, 10 mL / L 1 M MgS0 2 , 10 mL / L 2 M glucose) is added to the cells. The cells are cultivated in an overhead shaker for approx. 70 min at 37 ° C. The cells are then centrifuged by centrifugation at 7,000 g for 2 min at 4 ° C. The supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended. The cells are then spread on an LB agar plate preheated to 37 ° C. with 20 mg / L tetracycline and 20 mg / L chloramphenicol and incubated at 37 ° C. overnight.
2. Kultivierung und Induktion der Expression 2. Cultivation and induction of expression
Am nächsten Tag wird eine einzelne Kolonie gepickt und zur Inokulation von 30 mL LB-Medium (10 g/L NaCl, 10 g/L Sojapepton, 5 g/L Hefeextrakt, pH 7,4) mit 20 mg/L Tetracyclin und 20 mg/L Chloramphenicol in einem 300 mL Schüttei kolben mit Schikanen genutzt. Die Kultur wird
bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler über Nacht kultiviert. The next day, a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 20 mg / L tetracycline and 20 mg / L Chloramphenicol used in a 300 mL Schüttei flask with baffles. The culture will cultivated overnight at 37 ° C. and 120 revolutions per minute (deflection 25 mm) on an orbital shaker.
Am darauf folgenden Tag werden 200 mL TB-Medium (4 g/L Glycerin, 12 g/L Sojapepton, 24 g/L Hefeextrakt, 2,31 g/L KH2PO4, 12,54 g/L K2HPO4, pH 7,4) mit 25 mg/L Tetracyclin in einem 1 L Schüttelkolben mit Schikanen unter Verwendung der Vorkultur auf eine optische Dichte von OD600 = 0,1 angeimpft und bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler kultiviert. Für die Expression im Schüttelkolben wird bei einer optischen Dichte von OD600 = 2-3 mit 0,1 mM IPTG induziert und die Kultivierung bei 37 °C fortgesetzt, bevor nach 20-22 h die Kultivierung durch Zellemte abgebrochen wird. The following day, 200 mL TB medium (4 g / L glycerine, 12 g / L soy peptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, pH 7.4) inoculated with 25 mg / L tetracycline in a 1 L shake flask with baffles using the preculture to an optical density of OD600 = 0.1 and cultivated at 37 ° C and 120 revolutions per minute (deflection 25 mm) on an orbital shaker. For expression in the shake flask, induction is carried out with 0.1 mM IPTG at an optical density of OD600 = 2-3 and cultivation is continued at 37 ° C., before cultivation is terminated by cells after 20-22 h.
Beispiel 3: Example 3:
Sekretorische Expression einer Kollagenpeptid-Komponente in E. coli mittels der katalytischenSecretory expression of a collagen peptide component in E. coli by means of the catalytic
Domäne einer Cellulase aus Bacillus subtilis KSM-64 Domain of a cellulase from Bacillus subtilis KSM-64
1. Transformation der Zellen 1. Transformation of the cells
Für jede Expression wird 1 pL des Vektors pET28 (50-100 ng/pL) mit einem pBR322-ori, umfassend ein Kanamycin-Resi stenzgen und eine der SEQ ID Nos. 22, zu 50 pL auf Eis aufgetauten, chemisch kompetenten E. coli BL21(DE3) gegeben und für 30 min auf Eis gelagert. Anschließend erfolgt ein Hitzeschock für 30 s bei 42 °C, woraufhin die Zellen direkt für 5 min auf Eis gekühlt werden, bevor 950 pL SOC-Medium (20 g/L Sojapepton, 5 g/L Hefeextrakt, 0,6 g/L NaCl, 0,2 g/L KCl, 10 mL/L 1 M MgCh, 10 mL/L 1 M MgS02, 10 mL/L 2 M Glukose) zu den Zellen gegeben wird. Die Zellen werden in einem Überkopfschüttler für ca. 70 min bei 37 °C kultiviert. Anschließend werden die Zellen durch Zentrifugation bei 7.000 g für 2 min bei 4 °C zentrifugiert. Der Überstand wird bis auf ca. 100 pL, in denen das Zellpellet resuspendiert wird, verworfen. Die Zellen werden anschließend auf einer bei 37 °C vorgewärmten LB-Agarplatte mit 100 mg/L Kanamycin ausgestrichen und über Nacht bei 37 °C bebrütet. For each expression, 1 pL of the vector pET28 (50-100 ng / pL) with a pBR322 ori comprising a kanamycin resistance gene and one of SEQ ID Nos. 22 chemically competent E. coli BL21 (DE3) thawed on ice to 50 pL and stored on ice for 30 min. This is followed by a heat shock for 30 s at 42 ° C., after which the cells are cooled directly on ice for 5 min before using 950 μl of SOC medium (20 g / L soy peptone, 5 g / L yeast extract, 0.6 g / L NaCl , 0.2 g / L KCl, 10 mL / L 1 M MgCh, 10 mL / L 1 M MgS0 2 , 10 mL / L 2 M glucose) is added to the cells. The cells are cultivated in an overhead shaker for approx. 70 min at 37 ° C. The cells are then centrifuged by centrifugation at 7,000 g for 2 min at 4 ° C. The supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended. The cells are then spread with 100 mg / L kanamycin on an LB agar plate preheated to 37 ° C. and incubated at 37 ° C. overnight.
2. Kultivierung und Induktion der Expression 2. Cultivation and induction of expression
Am nächsten Tag wird eine einzelne Kolonie gepickt und zur Inokulation von 30 mL LB-Medium (10 g/L NaCl, 10 g/L Sojapepton, 5 g/L Hefeextrakt, pH 7,4) mit 100 mg/L Kanamycin in einem
300 mL Schüttelkolben mit Schikanen genutzt. Die Kultur wird bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler über Nacht kultiviert. The next day, a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 100 mg / L Kanamycin in one 300 mL shake flask with baffles used. The culture is cultivated overnight on an orbital shaker at 37 ° C. and 120 revolutions per minute (deflection 25 mm).
Am darauf folgenden Tag werden 200 mL TB-Medium (4 g/L Glycerin, 12 g/L Sojapepton, 24 g/L Hefeextrakt, 2,31 g/L KH2PO4, 12,54 g/L K2HPO4, pH 7,4) mit 100 mg/L Kanamycin in einem 1 L-Schüttelkolben mit Schikanen unter Verwendung der Vorkultur auf eine optische Dichte von OD600 = 0,1 angeimpft und bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler kultiviert. Für die Expression im Schüttelkolben wird bei einer optischen Dichte von OD600 = 2-3 mit 0,1 mM IPTG induziert und die Kultivierung bei 37 °C fortgesetzt, bevor nach 20-22 h die Kultivierung durch Zellemte abgebrochen wird. The following day, 200 mL TB medium (4 g / L glycerine, 12 g / L soy peptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, pH 7.4) inoculated with 100 mg / L kanamycin in a 1 L shake flask with baffles using the preculture to an optical density of OD600 = 0.1 and cultivated at 37 ° C and 120 revolutions per minute (deflection 25 mm) on an orbital shaker. For expression in the shake flask, induction is carried out with 0.1 mM IPTG at an optical density of OD600 = 2-3 and cultivation is continued at 37 ° C., before cultivation is terminated by cells after 20-22 h.
Erfolgt die Expression nicht im Schüttelkolben sondern erst im Fermenter, so wird diese zuvor angesetzte Kultur nicht induziert und über Nacht bei 37 °C kultiviert und als die Vorkultur 2. Stufe verwendet. If the expression does not take place in the shake flask but only in the fermenter, this previously set culture is not induced and cultivated overnight at 37 ° C. and used as the second stage preculture.
Am nächsten Tag wird ein Bioreaktor mit 12 L TB-Medium (4 g/L Glycerin, 12 g/L Sojapepton, 24 g/L Hefeextrakt, 2,31 g/L KH2PO4, 12,54 g/L K2HPO4, pH 7,4) und 100 mg/L Kanamycin mit der Vorkultur 2. Stufe auf eine OD600 = von ca. 0,1 eingestellt. Die Rührerdrehfrequenz startet bei 200 Umdrehungen pro Minute und steigt entsprechend einer Rührkaskade bei Unterschreitung des relativen pCh-Werts von 60 % um jeweils 2 % bis die maximale Rührerdrehfrequenz von 1500 Umdrehungen pro Minute erreicht wird. Bei Unterschreiten des relativen pCL-Wertes von 30 % erfolgte eine Zufütterung des Zulaufes (insgesamt 3 L Volumen bestehend aus 600 g/L Glycerin, 90 g/L Hefeextrakt, 2 g/L MgCU7H?C)). Zur Begasung werden 12 Normliter Luft pro Minute verwendet und ein Druck von 0,2 bar eingestellt. Der pH-Wert wird mit 10 %-iger Phosphorsäure und 4 M Natriumhydroxid-Lösung konstant auf 7,0 gehalten. Die Kultivierung erfolgt bei 37 °C. Bei einer optischen Dichte von OD600 = 9,0-10,0 wird mit 0,1 mM IPTG induziert. Die Kultivierung wird nach Erreichen der stationären Phase beendet. The next day, a bioreactor with 12 L TB medium (4 g / L glycerine, 12 g / L soy peptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, pH 7, 4) and 100 mg / L kanamycin with the preculture 2nd stage adjusted to an OD600 = of approx. 0.1. The stirrer rotation frequency starts at 200 revolutions per minute and increases according to a stirring cascade when the relative pCh value falls below 60% by 2% each time until the maximum stirrer rotation frequency of 1500 revolutions per minute is reached. When the relative pCL value fell below 30%, the feed was fed (a total of 3 L volume consisting of 600 g / L glycerol, 90 g / L yeast extract, 2 g / L MgCU7H? C)). For gassing, 12 standard liters of air per minute are used and a pressure of 0.2 bar is set. The pH is kept constant at 7.0 with 10% phosphoric acid and 4 M sodium hydroxide solution. Cultivation takes place at 37 ° C. At an optical density of OD600 = 9.0-10.0, 0.1 mM IPTG is induced. The cultivation is ended after the stationary phase has been reached.
Beispiel 4: Example 4:
Gewinnung hydroxylierter rekombinanter Kollagenpeptid-Komponenten mittels E. coli Recovery of hydroxylated recombinant collagen peptide components using E. coli
1. Post-translationale Prolinhydroxylierung
Zur post-translationen Hydroxylierung von Kollagenpeptid-Komponenten wurden in verschiedenen Ansätzen der Vektor pACYCDuet-1, umfassend ein Chloramphenicol- Resistenzgen und die SEQ ID No. 23, bzw. der Vektor pCDFDuet-1, umfassend ein Streptomycin- Resistenzgen und die SEQ ID No. 23, in eine Kollagenpeptid-Komponente exprimierende E. coli Zellen transformiert. 1. Post-translational proline hydroxylation For post-translation hydroxylation of collagen peptide components, the vector pACYCDuet-1, comprising a chloramphenicol resistance gene and SEQ ID NO. 23, or the vector pCDFDuet-1, comprising a streptomycin resistance gene and SEQ ID no. 23, transformed into E. coli cells expressing a collagen peptide component.
Je nach gewählter Kollagenpeptid-Komponente kann durch Co-Expression mit einer bevorzugt in Y-Position des Kollagenmotivs (Gly-X-Y)n hydroxylierenden Prolyl-4-hydroxylase, wie der Prolyl-4-hydroxylase 1 aus Arabidopsis thaliana , sowohl die cytosolische Expression von hydroxylierten Kollagen-Komponenten (vgl. Beispiel 1) als auch die sekretorische Expression von hydroxylierten Kollagen-Komponenten (vgl. Beispiele 2 und 3) durch E. coli Zellen realisiert werden. Massenspektrometrisch konnte die post-translationale Hydroxylierung von Prolin nachgewiesen werden. Depending on the selected collagen peptide component of the collagenous motif (Gly-XY) both the cytosolic expression can be obtained by co-expression with a preferably Y-position n hydroxylating prolyl hydroxylase 4-how of prolyl hydroxylase 4-1 from Arabidopsis thaliana, of hydroxylated collagen components (see. Example 1) and the secretory expression of hydroxylated collagen components (see. Examples 2 and 3) can be realized by E. coli cells. The post-translational hydroxylation of proline could be demonstrated by mass spectrometry.
1.1 Bestimmung des Hydroxylierungsgrades 1.1 Determination of the degree of hydroxylation
Die Bestimmung des Hydroxylierungsgrades basierte auf der Hydroylse der zu analysierenden Proteinprobe in die einzelnen Aminosäuren, deren Derivatisierung (AQC-Reagenz) und der anschließenden Auftrennung mittels analytischer Hochleistungsflüssigkeits-Chromatographie (HPLC). Aus dem Chromatogramm wurde dann unter Zuhilfenahme eines Prolin- und Hy droxyprolin- Standards aus den Peakflächen von Prolin und Hydroxyprolin aus der zu analysierenden Probe der Hydroxylierungsgrad berechnet, wobei der Hydroxylierungsgrad der Anteil hydroxylierter Prolinreste (Hydroxyprolin) bezogen auf die molare Summe aller Proline (Hydroxyprolin und Prolin) ist (Mol Hydroxyprolin/(Mol Hydroxyprolin + Mol Prolin). The determination of the degree of hydroxylation was based on the hydrolysis of the protein sample to be analyzed into the individual amino acids, their derivatization (AQC reagent) and the subsequent separation by means of analytical high-performance liquid chromatography (HPLC). The degree of hydroxylation was then calculated from the chromatogram with the aid of a proline and hydroxyproline standard from the peak areas of proline and hydroxyproline from the sample to be analyzed, the degree of hydroxylation being the proportion of hydroxylated proline residues (hydroxyproline) based on the molar sum of all proline (hydroxyproline and proline) is (mole hydroxyproline / (mole hydroxyproline + mole proline).
Die zu analysierenden aufgereinigten Kollagenpeptide wurden für >24 h bei 110°C in 6 M Salzsäure (Endkonzentration) bei einer Proteinbeladung von 10 g/L in geschlossenen Reaktionsgefäßen hydrolysiert. Nach Abkühlen auf Eis, tropfenweisem Neutralisieren auf Eis mit Proben-identischem Volumen 6 M Natronlauge, Abzentrifugieren der Proben für 5 min bei 13.000 min 1 in einer Mikroliterzentrifuge und ggf. Verdünnen der Proben mit 0,2 M Natriumborat-Puffer pH 9,0 erfolgt die Derivatisierung. Hierzu wurden 100 pL Probe mit 700 pL 0,2 M Natriumborat- Puffer pH 9,0 und 200 pL AQC-Reagenz (2 mg/mL 6-Aminoqinolyl-N-hydroxysuccinimidyl carbamate in Acetonitril (p.A.) bei 55°C gelöst) in geschlossenen HPLC-Gefäßen gemischt und im Wasserbad bei 55 °C für 10 min inkubiert. Anschließend erfolgte die analytische Auftrennung mittels Hochleistungsflüssigkeits-Chromatographie (Knauer 250 mm x 4 mm; Eurospher II 1005
C18P; Säulentemperatur: 37 °C; Probenschleife: 20 pL; Volumenstrom: 0,8 bis 1,0 mL/min; Fluoreszenzdetektor (Shimadzu RF-551)): 250 nm/395 nm; Gain: x32; Sensitivity: low) unter Verwendung des folgenden Puffer-Gradienten aus Puffer A (95% (v/v) 0,3 M Natriumacetat pH 6,5 + 5% (v/v) Acetonitril) und Puffer B (20% (v/v) Acetonitril + 60% (v/v) Methanol + 20% (v/v). The purified collagen peptides to be analyzed were hydrolyzed for> 24 h at 110 ° C. in 6 M hydrochloric acid (final concentration) with a protein load of 10 g / L in closed reaction vessels. After cooling on ice, neutralizing drop by drop on ice with sample-identical volume 6 M sodium hydroxide solution, centrifuging the samples for 5 min at 13,000 min 1 in a microliter centrifuge and, if necessary, diluting the samples with 0.2 M sodium borate buffer pH 9.0 the derivatization. For this purpose, 100 pL sample with 700 pL 0.2 M sodium borate buffer pH 9.0 and 200 pL AQC reagent (2 mg / mL 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate dissolved in acetonitrile (pA) at 55 ° C) in closed HPLC vessels mixed and incubated in a water bath at 55 ° C for 10 min. The analytical separation then took place by means of high-performance liquid chromatography (Knauer 250 mm x 4 mm; Eurospher II 1005 C18P; Column temperature: 37 ° C; Sample loop: 20 pL; Volume flow: 0.8 to 1.0 mL / min; Fluorescence detector (Shimadzu RF-551): 250 nm / 395 nm; Gain: x32; Sensitivity: low) using the following buffer gradient from buffer A (95% (v / v) 0.3 M sodium acetate pH 6.5 + 5% (v / v) acetonitrile) and buffer B (20% (v / v) v) acetonitrile + 60% (v / v) methanol + 20% (v / v).
1.2 Hydroxylierungsgrad nach post-translationaler Hydroxylierung 1.2 Degree of hydroxylation after post-translational hydroxylation
Bei der post-translationalen Hydroxylierung mittels Prolyl-4-hydroxylase (P4H) ist davon auszugehen, dass diese weitestgehend spezifisch ausschließlich Prolinreste in der rekombinanten Kollagensequenz hydroxyliert. Demzufolge tragen etwaige in der Probe enthaltene Proteinverunreinigungen ausschließlich zur gemessenen Peakfläche des Prolinpeaks bei und vergrößern somit den Nenner bei der Berechnung des Hydroxylierungsgrades. Folglich führen Proteinverunreinigungen zu einer Verringerung des gemessenen Hydroxylierungsgrades, welcher bezogen auf hochreines Kollagenpeptid höher ausfallen würde. Die ermittelten Hydroxylierungsgrade der Kollagenpeptide stellen im Falle der post-translationalen Hydroxylierung demnach Mindestwerte dar. In the case of post-translational hydroxylation by means of prolyl 4-hydroxylase (P4H), it can be assumed that this hydroxylates exclusively proline residues in the recombinant collagen sequence specifically as far as possible. As a result, any protein impurities contained in the sample only contribute to the measured peak area of the proline peak and thus increase the denominator when calculating the degree of hydroxylation. Consequently, protein impurities lead to a reduction in the measured degree of hydroxylation, which would be higher in relation to highly pure collagen peptide. The determined degrees of hydroxylation of the collagen peptides represent minimum values in the case of post-translational hydroxylation.
1.3 Post-translationale Hydroxylierung eines CD-Cel-TEV- 10er -Fusionpeptids mittels At- P4H 1.3 Post-translational hydroxylation of a CD-Cel-TEV-10 fusion peptide using At-P4H
Bei der Co-Expression des 10 kDa Kollagenfragmentes in Fusion mit der katalytischen Domäne der Cellulase aus Bacillus subtilis KSM-64 (CD-Cel-TEV-10er; siehe Beispiel 3) und der P4H aus Arabidopsis thaliana (4/-P4H) konnte in einem Zulaufverfahren mit anschließender Ni-NTA- Affinitätschromatographie reproduzierbar ein Hydroxylierungsgrad im Bereich von mindestens 22-26 % gemessen werden. Der gemessene Hydroxylierungsgrad bezieht sich auf das Fusionsprotein, welches in seiner verkürzten Form 41 Prolinreste enthält, von denen 15 Prolinreste in der verkürzten Cellulase-Domäne und 26 Prolinreste in dem Kollagenfragment enthalten sind. Da davon auszugehen ist, dass die 15 Prolinreste der Cellulase-Domäne nicht von der co- exprimierten At- P4H hydroxyliert werden, denn dieser Teil entspricht nicht dem natürlichen At- P4H-Substratspektrum, ist der tatsächliche Hydroxylierungsgrad der rekombinanten Kollagensequenz höher ist als der Hydroxylierungsgrad des Fusionsproteins. In the co-expression of the 10 kDa collagen fragment in fusion with the catalytic domain of the cellulase from Bacillus subtilis KSM-64 (CD-Cel-TEV-10er; see Example 3) and the P4H from Arabidopsis thaliana (4 / -P4H), in a feed process with subsequent Ni-NTA affinity chromatography can reproducibly measure a degree of hydroxylation in the range of at least 22-26%. The measured degree of hydroxylation relates to the fusion protein, which in its truncated form contains 41 proline residues, of which 15 are proline residues in the truncated cellulase domain and 26 proline residues in the collagen fragment. Since it can be assumed that the 15 proline residues of the cellulase domain are not hydroxylated by the co-expressed At-P4H, because this part does not correspond to the natural At-P4H substrate spectrum, the actual degree of hydroxylation of the recombinant collagen sequence is higher than the degree of hydroxylation of the fusion protein.
2. Prä-translationale Prolinhydroxylierung
Zur prä-translationalen Hydroxylierung von Kollagenpeptid-Komponenten wurden Kollagenpeptid-Komponente exp dinierende K. coli Zellen mit einem Vektor, umfassend die SEQ ID No. 24 transformiert, um eine Co-Expression von zu hydroxylierenden Kollagenpeptid- Komponenten und einer Prolin-4-hydroxylase (PIN4H) aus Streptomyces griseoviridis zu erzielen. Massenspektroskopisch konnte der Einbau von Hydroxyprolin in die exprimierten Kollagenpeptid-Komponenten nachgewiesen werden. 2. Pre-translational proline hydroxylation For the pre-translational hydroxylation of collagen peptide components, collagen peptide component exp dinating K. coli cells were combined with a vector comprising SEQ ID NO. 24 transformed in order to achieve a co-expression of collagen peptide components to be hydroxylated and a proline-4-hydroxylase (PIN4H) from Streptomyces griseoviridis. The incorporation of hydroxyproline into the expressed collagen peptide components could be demonstrated by mass spectroscopy.
2.1 Bestimmung des Hydroxylierungsgrades 2.1 Determination of the degree of hydroxylation
Die Bestimmung des Hydroxylierungsgrades erfolgte wie im vorstehenden Absatz 1.1 beschrieben. The degree of hydroxylation was determined as described in paragraph 1.1 above.
2.2 Hydroxylierungsgrad nach prä-translationaler Hydroxylierung 2.2 Degree of hydroxylation after pre-translational hydroxylation
Im Gegensatz zur post-translationen Hydroxylierung ist der prä-translationale Hydroxylierungsansatz unter Einsatz einer Prolin-4-hydroxylase (PIN4H) unspezifisch, d.h. dass der Einbau von Hydroxyprolin an Stelle von Prolin während der Translation zufällig erfolgt und im Wesentlichen von der Verfügbarkeit der mit Hydroxyprolin-beladenen tRNA abhängt. Da die Co-Expression der PIN4H und der Kollagenpeptide erst nach Aufbau von ausreichend proteinhaltiger Biomasse im Rahmen der Fermentation erfolgt, ist davon auszugehen, dass ein gewisser Anteil an Wirtsproteinen, die als Kontaminanten in der zu analysierenden Proteinprobe vorhanden sein können, nicht-hydroxyliert vorliegen und der ermittelte Hydroxylierungsgrad geringer ist als dies beim Vorliegen eines hochreinen Kollagenpeptids der Fall wäre. Daher ist davon auszugehen, dass die im Falle der post-translationalen Hydroxylierung ermittelten Hydroxylierungsgrade Mindestwerte darstellen. In contrast to post-translation hydroxylation, the pre-translational hydroxylation approach using a proline-4-hydroxylase (PIN4H) is unspecific, ie the incorporation of hydroxyproline instead of proline occurs randomly during translation and essentially depends on the availability of hydroxyproline -loaded tRNA. Since the co-expression of PIN4H and the collagen peptides only takes place after a sufficient protein-containing biomass has been built up during fermentation, it can be assumed that a certain proportion of host proteins, which may be present as contaminants in the protein sample to be analyzed, are present in non-hydroxylated form and the determined degree of hydroxylation is less than would be the case in the presence of a highly pure collagen peptide. It can therefore be assumed that the degrees of hydroxylation determined in the case of post-translational hydroxylation represent minimum values.
2.3 Prä-translationale Hydroxylierung eines CD-Cel-TEV-1 Oer-Fusionpeptids mittels Sg- PIN4H 2.3 Pre-translational hydroxylation of a CD-Cel-TEV-1 Oer fusion peptide using Sg-PIN4H
Bei der Co-Expression des 10 kDa Kollagenfragmentes in Fusion mit der katalytischen Domäne der Cellulase aus Bacillus subtilis KSM-64 (CD-Cel-TEV-1 Oer; siehe Beispiel 3) und der PIN4H aus Streptomyces sriseoviridis (L' -RIN4H) (SEQ ID No. 10) konnte in einem Zulaufverfahren mit anschließender Ni-NTA-Affinitätschromatographie ein Hydroxylierungsgrad des Fusionspeptids im Bereich von mindestens 10-14 % gemessen werden. In the co-expression of the 10 kDa collagen fragment in fusion with the catalytic domain of the cellulase from Bacillus subtilis KSM-64 (CD-Cel-TEV-1 Oer; see Example 3) and the PIN4H from Streptomyces sriseoviridis (L '-RIN4H) ( SEQ ID No. 10) it was possible to measure a degree of hydroxylation of the fusion peptide in the range of at least 10-14% in a feed process with subsequent Ni-NTA affinity chromatography.
Beispiel 5:
Cvtosolische Expression einer hydroxylierten Kollagenpeptid-Komponente in E. coli Example 5: Cytosolic expression of a hydroxylated collagen peptide component in E. coli
1. Transformation der Zellen 1. Transformation of the cells
Für jede Expression wird 1 pL des Vektor pET-28a(+)-Vektor (50-100 ng/pL) umfassend ein Kanamycin-Resi stenzgen und eine Nukleinsäuresequenz gemäß SEQ ID No. 32, die das Kollagenpeptid gemäß SEQ ID No. 33 codiert, und 1 pL eines Expressionsplasmids, welches entweder eine At- P4H codierende Sequenz (pACYCDuet-1) oder eine L#-RIN4H codierende Sequenz (pCDFDuet-1) aufweist, zu 50 pL auf Eis aufgetauten, chemisch kompetenten E. coli BL21 gegeben und für 30 min auf Eis gelagert. Anschließend erfolgt ein Hitzeschock für 30 s bei 42 °C, woraufhin die Zellen direkt für 5 min auf Eis gekühlt werden, bevor 950 pL SOC-Medium (20 g/L Sojapepton, 5 g/L Hefeextrakt, 0,6 g/L NaCl, 0,2 g/L KCl, 10 mL/L 1 M MgCh, 10 mL/L 1 M MgSCE, 10 mL/L 2 M Glukose) zu den Zellen gegeben wird. Die Zellen werden in einem Überkopfschüttler für ca. 70 min bei 37 °C kultiviert. Anschließend werden die Zellen durch Zentrifugation bei 7.000 g für 2 min bei 4 °C zentrifugiert. Der Überstand wird bis auf ca. 100 pL, in denen das Zellpellet resuspendiert wird, verworfen. Die Zellen werden anschließend auf einer bei 37 °C vorgewärmten LB Agarplatte mit 80 mg/L Kanamycin und 20 mg/L Chloramphenicol (P4H) beziehungsweise 20 mg/L Streptomycin (PIN4H) ausgestrichen und über Nacht bei 37 °C bebrütet. For each expression, 1 pL of the vector pET-28a (+) - vector (50-100 ng / pL) comprising a kanamycin resistance gene and a nucleic acid sequence according to SEQ ID no. 32, which contain the collagen peptide according to SEQ ID no. 33, and 1 pL of an expression plasmid which has either an At-P4H coding sequence (pACYCDuet-1) or an L # -RIN4H coding sequence (pCDFDuet-1) to 50 pL, chemically competent E. coli BL21, thawed on ice given and stored on ice for 30 min. This is followed by a heat shock for 30 s at 42 ° C., after which the cells are cooled directly on ice for 5 min before using 950 μl of SOC medium (20 g / L soy peptone, 5 g / L yeast extract, 0.6 g / L NaCl , 0.2 g / L KCl, 10 mL / L 1 M MgCh, 10 mL / L 1 M MgSCE, 10 mL / L 2 M glucose) is added to the cells. The cells are cultivated in an overhead shaker for approx. 70 min at 37 ° C. The cells are then centrifuged by centrifugation at 7,000 g for 2 min at 4 ° C. The supernatant is discarded up to approx. 100 pL, in which the cell pellet is resuspended. The cells are then spread on an LB agar plate preheated to 37 ° C. with 80 mg / L kanamycin and 20 mg / L chloramphenicol (P4H) or 20 mg / L streptomycin (PIN4H) and incubated at 37 ° C. overnight.
2. Kultivierung und Induktion der Expression 2. Cultivation and induction of expression
Am nächsten Tag wird eine einzelne Kolonie gepickt und zur Inokulation von 30 mL LB-Medium (10 g/L NaCl, 10 g/L Sojapepton, 5 g/L Hefeextrakt, pH 7,4) mit 80 mg/L Kanamycin und 20 mg/L Chloramphenicol (P4H) beziehungsweise 20 mg/L Streptomycin (PIN4H) in einem 300 mL Schüttelkolben mit Schikanen genutzt. Die Kultur wird bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler über Nacht kultiviert. The next day a single colony is picked and for inoculation of 30 mL LB medium (10 g / L NaCl, 10 g / L soy peptone, 5 g / L yeast extract, pH 7.4) with 80 mg / L kanamycin and 20 mg / L chloramphenicol (P4H) or 20 mg / L streptomycin (PIN4H) in a 300 mL shake flask with baffles. The culture is cultivated overnight on an orbital shaker at 37 ° C. and 120 revolutions per minute (deflection 25 mm).
Am darauf folgenden Tag werden 200 mL TB-Medium (4 g/L Glycerin, 12 g/L Sojapepton, 24 g/L Hefeextrakt, 2,31 g/L KH2PO4, 12,54 g/L K2HPO4, pH 7,4) mit 80 mg/L Kanamycin und 20 mg/L Chloramphenicol (P4H) beziehungsweise 20 mg/L Streptomycin (PIN4H) in einem 1 L Schüttelkolben mit Schikanen unter Verwendung der Vorkultur auf eine optische Dichte von OD600 = 0,1 angeimpft und bei 37 °C und 120 Umdrehungen pro Minute (Auslenkung 25 mm) auf einem Orbitalschüttler kultiviert. Für die Expression im Schüttelkolben wird bei einer
optischen Dichte von OD600 = 2-3 mit 0,1 mM IPTG induziert und die Kultivierung bei 37 °C fortgesetzt, bevor nach 20-22 h die Kultivierung durch Zellemte abgebrochen wird. The following day, 200 mL TB medium (4 g / L glycerine, 12 g / L soy peptone, 24 g / L yeast extract, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, pH 7.4) with 80 mg / L kanamycin and 20 mg / L chloramphenicol (P4H) or 20 mg / L streptomycin (PIN4H) in a 1 L shake flask with baffles using the preculture to an optical density of OD600 = 0.1 and inoculated at 37 ° C and 120 revolutions per minute (deflection 25 mm) cultivated on an orbital shaker. For expression in the shake flask, a optical density of OD600 = 2-3 is induced with 0.1 mM IPTG and cultivation is continued at 37 ° C. before cultivation is terminated by cell harvesting after 20-22 h.
Erfolgte die Expression nicht im Schüttelkolben, sondern erst im Fermenter, so wird diese zuvor angesetzte Kultur nicht induziert und über Nacht bei 37 °C kultiviert und als Vorkultur verwendet. Am nächsten Tag wird ein Bioreaktor (19 L NLF, Bioengineering AG) mit 12 L TB-Medium und 80 mg/L Kanamycin und 20 mg/L Chloramphenicol (P4H) beziehungsweise 20 mg/L Streptomycin (PIN4H) mit der Vorkultur auf eine OD600 von ca. 0,1 eingestellt. Die Rührerdrehfrequenz startet bei 200 Umdrehungen pro Minute und steigt entsprechend einer Rührkaskade bei Unterschreitung des relativen pCb-Werts von 30 % um jeweils 2 %. Die maximale Rührerdrehfrequenz von 1500 Umdrehungen pro Minute wird dabei nicht erreicht. Bei Überschreiten des relativen pCb-Werts von 60 % erfolgte so lange eine Zufütterung des Zulaufes, bis der relative pCk-Wert wieder unter 60% liegt (insgesamt 3 L Volumen bestehend aus 600 g/L Glycerin, 90 g/L Hefeextrakt, 2 g/L MgCbUHiO). Zur Begasung werden 15 Normliter Luft pro Minute verwendet und ein Überdruck von 0,2 bar eingestellt. Der pH-Wert wird mit 10 %iger (w/w) Phosphorsäure und 25% (w/w) Ammoniakwasser konstant auf 7,0 gehalten. AlsIf the expression did not take place in the shake flask but only in the fermenter, this previously set culture is not induced and cultivated overnight at 37 ° C. and used as a preculture. The next day, a bioreactor (19 L NLF, Bioengineering AG) with 12 L TB medium and 80 mg / L kanamycin and 20 mg / L chloramphenicol (P4H) or 20 mg / L streptomycin (PIN4H) with the preculture is adjusted to an OD600 set from approx. 0.1. The stirrer rotation frequency starts at 200 revolutions per minute and increases by 2% in each case in accordance with a stirring cascade when the relative pCb value falls below 30%. The maximum stirrer rotation frequency of 1500 revolutions per minute is not reached. If the relative pCb value of 60% was exceeded, the feed was added until the relative pCb value is below 60% again (a total of 3 L volume consisting of 600 g / L glycerol, 90 g / L yeast extract, 2 g / L MgCbUHiO). 15 standard liters of air per minute are used for gassing and an overpressure of 0.2 bar is set. The pH value is kept constant at 7.0 with 10% (w / w) phosphoric acid and 25% (w / w) ammonia water. as
Anti schaummittel wird Pluronic® PE 8100 verwendet. Die Kultivierung erfolgt bei 37 °C. Bei einer optischen Dichte von OD600 von 9,0-10,0 wird mit 0,1 mM IPTG induziert. Die Kultivierung wird nach Erreichen der stationären Phase beendet.
Pluronic® PE 8100 is used as an anti-foaming agent. Cultivation takes place at 37 ° C. At an optical density of OD600 of 9.0-10.0, 0.1 mM IPTG is induced. The cultivation is ended after the stationary phase has been reached.
Claims
1. Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid- Komponente in prokaryoti sehen Systemen, umfassend die Schritte: a) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolyl-4-hydroxylase kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst, b) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolyl-4-hydroxylase zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, c) Gewinnen der hydroxylierten Kollagenpeptid-Komponente, wobei das mindestens eine Kollagenpeptid das Aminosäuresequenzmotiv (Gly-X-Y)n aufweist und mindestens 55% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. 1. A method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: a) providing a prokaryotic see expression system comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one coding for at least one prolyl-4-hydroxylase Nucleotide sequence, wherein the nucleotide sequence coding for at least one recombinant collagen peptide component comprises the nucleotide sequence of at least one collagen peptide, b) cultivating the prokaryotic expression system in a culture medium under conditions that allow the expression of the at least one recombinant collagen peptide component and the at least one prolyl-4- allow hydroxylase to obtain at least one hydroxylated collagen peptide component, the collagen peptide component encoding the at least one nucleotide sequence encoding the collagen peptide component en collagen peptide has a reduced hydrophobicity and / or a reduced proline content, c) obtaining the hydroxylated collagen peptide component, the at least one collagen peptide having the amino acid sequence motif (Gly-XY) n and at least 55% of the hydroxylations in the at least one collagen peptide on a proline are in the Y position.
2. Verfahren nach Anspruch 1, wobei in der die Kollagenpeptid-Komponente kodierenden Nukleotidsequenz des prokaryoti sehen Expressionssystems die ein Kollagenpeptid kodierende Nukleotidsequenz mit mindestens einer Nukleotidsequenz fusioniert ist, die einen Peptidrest kodiert. 2. The method according to claim 1, wherein in the nucleotide sequence of the prokaryotic expression system coding for the collagen peptide component, the nucleotide sequence coding for a collagen peptide is fused with at least one nucleotide sequence which codes for a peptide residue.
3. Verfahren nach Anspruch 2, wobei der Peptidrest mindestens einen Protein-tag, ein Signalpeptid und/oder eine Kaschierungsdomäne aufweist.
3. The method according to claim 2, wherein the peptide residue has at least one protein tag, a signal peptide and / or a lamination domain.
4. Verfahren nach einem der Ansprüche 2 und 3, wobei der Peptidrest abspaltbar ist, insbesondere enzymatisch abspaltbar. 4. The method according to any one of claims 2 and 3, wherein the peptide residue is cleavable, in particular enzymatically cleavable.
5. Verfahren nach einem der Ansprüche 2 bis 4, wobei nach Schritt c) oder nach Schritt b) und vor Schritt c) eine Abspaltung des mindestens einen Peptidrests der hydroxylierten Kollagenpeptid-Komponente erfolgt. 5. The method according to any one of claims 2 to 4, wherein after step c) or after step b) and before step c) there is cleavage of the at least one peptide residue of the hydroxylated collagen peptide component.
6. Verfahren nach einem der Ansprüche 2 bis 5, wobei die hydroxylierte Kollagenpeptid- Komponente ein Kollagenpeptid und mindestens ein N- und/oder C-terminales Sekretions- Signalpeptid umfasst. 6. The method according to any one of claims 2 to 5, wherein the hydroxylated collagen peptide component comprises a collagen peptide and at least one N- and / or C-terminal secretion signal peptide.
7. Verfahren nach einem der Ansprüche 1 bis 6, wobei das durch die Nukleotidsequenz kodierte Kollagenpeptid eine in bovinem Kollagen, insbesondere in bovinem Typ I Kollagen, bevorzugt in der al -Kette des bovinen Typ I Kollagen, vorkommende Aminosäuresequenz aufweist. 7. The method according to any one of claims 1 to 6, wherein the collagen peptide encoded by the nucleotide sequence has an amino acid sequence occurring in bovine collagen, in particular in bovine type I collagen, preferably in the a1 chain of bovine type I collagen.
8. Verfahren nach einem der Ansprüche 1 bis 7, wobei mindestens 70% der Hydroxylierungen in dem mindestens einen Kollagenpeptid an einem Prolin in Y-Position vorliegen. 8. The method according to any one of claims 1 to 7, wherein at least 70% of the hydroxylations in the at least one collagen peptide are present on a proline in the Y position.
9. Verfahren nach einem der Ansprüche 1 bis 8, wobei es sich bei der Prolyl-4-hydroxylase kodierenden Nukleotidsequenz um eine Nukleotidsequenz aus Arabidopsis thaliana handelt. 9. The method according to any one of claims 1 to 8, wherein the nucleotide sequence coding for prolyl-4-hydroxylase is a nucleotide sequence from Arabidopsis thaliana.
10. Verfahren nach einem der Ansprüche 1 bis 8, wobei die Prolyl-4-hydroxylase eine Aminosäuresequenz gemäß SEQ ID No. 15 aufweist. 10. The method according to any one of claims 1 to 8, wherein the prolyl 4-hydroxylase has an amino acid sequence according to SEQ ID no. 15 has.
11. Verfahren zur Herstellung einer rekombinanten hydroxylierten Kollagenpeptid- Komponente in prokaryoti sehen Systemen, umfassend die Schritte: i) Bereitstellung eines prokaryoti sehen Expressionssystems, umfassend mindestens eine mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz und mindestens eine mindestens eine Prolin-4-hydroxylase (PIN4H) kodierende Nukleotidsequenz, wobei die mindestens eine rekombinante Kollagenpeptid-Komponente kodierende Nukleotidsequenz die Nukleotidsequenz mindestens eines Kollagenpeptids umfasst,
ii) Kultivieren des prokaryoti sehen Expressionssystems in einem Kulturmedium unter Bedingungen, die die Expression der mindestens einen rekombinanten Kollagenpeptid- Komponente und der mindestens einen Prolin-4-hydroxylase (PIN4H) zum Erhalt mindestens einer hydroxylierten Kollagenpeptid-Komponente ermöglichen, wobei die Kollagenpeptid- Komponente gegenüber dem mindestens einen von der Kollagenpeptid-Komponente kodierenden Nukleotidsequenz kodierten Kollagenpeptid eine verringerte Hydrophobizität und/oder einen verringerten Prolingehalt aufweist, iii) Gewinnen der hydroxylierten Kollagenpeptid-Komponente. 11. A method for producing a recombinant hydroxylated collagen peptide component in prokaryotic see systems, comprising the steps: i) providing a prokaryotic see expression system, comprising at least one nucleotide sequence coding for at least one recombinant collagen peptide component and at least one at least one proline-4-hydroxylase ( PIN4H) nucleotide sequence coding, wherein the nucleotide sequence coding for at least one recombinant collagen peptide component comprises the nucleotide sequence of at least one collagen peptide, ii) Cultivating the prokaryotic expression system in a culture medium under conditions which enable the expression of the at least one recombinant collagen peptide component and the at least one proline 4-hydroxylase (PIN4H) to obtain at least one hydroxylated collagen peptide component, the collagen peptide component has a reduced hydrophobicity and / or a reduced prolin content compared to the at least one nucleotide sequence encoded by the collagen peptide component, iii) recovering the hydroxylated collagen peptide component.
12. Verfahren nach Anspruch 11, wobei die mindestens eine Prolin-4-hydroxylase eine monomere Prolin-4-hydroxylase ist. 12. The method of claim 11, wherein the at least one proline-4-hydroxylase is a monomeric proline-4-hydroxylase.
13. Verfahren nach Anspruch 11 oder 12, wobei die mindestens eine Prolin-4-hydroxylase bakteriellen Ursprungs ist. 13. The method according to claim 11 or 12, wherein the at least one proline-4-hydroxylase is of bacterial origin.
14. Verfahren nach Anspruch 13, wobei die mindestens eine Prolin-4-hydroxylase eine Prolin- 4-hydroxylase aus Streptomyces griseoviridis, Dactylosporangium sp., Pseudomonas stutzeri, Bordetella bronchiseptica, Bradyrhizobium japonicum, Aeromonas caviae, Janthinobacterium sp. oder Achromobacter xylosoxidans ist. 14. The method according to claim 13, wherein the at least one proline 4-hydroxylase is a proline 4-hydroxylase from Streptomyces griseoviridis, Dactylosporangium sp., Pseudomonas stutzeri, Bordetella bronchiseptica, Bradyrhizobium japonicum, Aeromonas caviae, Janthinobacterium sp. or Achromobacter xylosoxidans.
15. Verfahren nach einem der Ansprüche 11 bis 14, wobei die mindestens eine Prolin-4- hydroxylase eine Aminosäuresequenz ausgewählt aus SEQ ID No. 10 bis 13 aufweist, bevorzugt aus einer Aminosäuresequenz ausgewählt aus SEQ ID No. 10 bis 13 besteht. 15. The method according to any one of claims 11 to 14, wherein the at least one proline-4-hydroxylase has an amino acid sequence selected from SEQ ID NO. 10 to 13, preferably from an amino acid sequence selected from SEQ ID NO. 10 to 13 consists.
16. Hydroxylierte Kollagenpeptid-Komponente, hergestellt durch ein Verfahren gemäß einem der Ansprüche 1 bis 10. 16. Hydroxylated collagen peptide component produced by a method according to any one of claims 1 to 10.
17. Hydroxylierte Kollagenpeptid-Komponente, hergestellt durch das Verfahren gemäß einem der Ansprüche 11 und 15.
17. Hydroxylated collagen peptide component produced by the method according to any one of claims 11 and 15.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102020205703.6A DE102020205703A1 (en) | 2020-05-06 | 2020-05-06 | Expression of collagen peptide components in prokaryotic systems |
| PCT/EP2021/061820 WO2021224316A1 (en) | 2020-05-06 | 2021-05-05 | Expression of collagen peptide components in procaryotic systems |
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| CN116813749B (en) * | 2023-06-13 | 2024-01-30 | 广州启点生物科技有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
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| CN109022464A (en) * | 2018-07-02 | 2018-12-18 | 西安巨子生物基因技术股份有限公司 | The hydroxylacion method of recombination human source collagen type |
| EP3853248A4 (en) * | 2018-08-17 | 2022-06-22 | Modern Meadow, Inc. | Fusion proteins for hydroxylating amino acids and products |
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