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CN-121986108-A - Recombinant proteins, recombinant DNA sequences, vectors, eukaryotic and prokaryotic expression systems and uses thereof

CN121986108ACN 121986108 ACN121986108 ACN 121986108ACN-121986108-A

Abstract

The present invention relates to recombinant RE15mR and EJ17zipR hybrid proteins having the structures described in the specification, recombinant DNA sequences encoding the recombinant hybrid proteins, vectors containing the recombinant DNA sequences, and eukaryotic or prokaryotic expression systems transformed with the recombinant DNA sequences. The invention also relates to the use of the recombinant hybrid protein as a component of a bio-ink intended for bio-printing and as a component of a biological material. Furthermore, the invention relates to the use of the recombinant DNA sequences, vectors and eukaryotic or prokaryotic expression systems for the production of recombinant hybrid proteins.

Inventors

  • M. Wozola
  • K. Kossoska
  • P. Koretzka
  • A. Romanok Khrushchev
  • V. Caichula Adamchevska
  • I. Sokolovska
  • N. Lukasievitz
  • M. CLARK
  • S. Domanski
  • K. Floris Yankovska
  • A. Zakhevska

Assignees

  • 波尔比奥尼卡股份有限公司
  • 科学研究与发展基金会

Dates

Publication Date
20260505
Application Date
20240307
Priority Date
20230712

Claims (20)

  1. 1. A recombinant RE15mR hybrid protein, it consists of 3 arthropod elastin domains, 1 K+ domain, 3 arthropod elastin domains, 1 MMP domain, 3 elastin domains, 1 K+ domain, 4 elastin domains 1 K+ domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 MMP domain, 7 RGD domain composition.
  2. 2. The recombinant RE15mR hybrid protein according to claim 1, wherein the amino acid sequence contains 12-15% proline and/or 32-38% glycine.
  3. 3. The recombinant RE15mR hybrid protein according to claim 1 or 2, wherein the protein has a molecular weight of 26kDa.
  4. 4. A recombinant RE15mR hybrid protein according to any one of claims 1-3 having the amino acid sequence of seq#3.
  5. 5. The recombinant RE15mR hybrid protein according to any one of claims 1-4, which constitutes a fragment of the recombinant protein.
  6. 6. The recombinant RE15mR hybrid protein according to any one of claims 1-5, which is at least partially methacrylated.
  7. 7.A recombinant DNA sequence selected from the group consisting of: A recombinant DNA sequence encoding a recombinant RE15mR hybrid protein as defined in any one of claims 1 to 4, A recombinant DNA sequence comprising a region comprising a DNA sequence encoding a recombinant RE15mR hybrid protein as defined in any one of claims 1-4, and A recombinant DNA sequence which hybridizes to a DNA sequence encoding a recombinant RE15mR hybrid protein as defined in any one of claims 1 to 4.
  8. 8. The recombinant DNA sequence according to claim 7, having a sequence at least 80% identical, more preferably at least 90% identical, most preferably homologous to the recombinant DNA sequence of seq#1.
  9. 9. A carrier, comprising Recombinant DNA sequence comprising a region comprising a recombinant DNA sequence as defined in claim 7 or 8, and/or A recombinant sequence comprising a portion of a region comprising a recombinant DNA sequence as defined in claim 7 or 8.
  10. 10. The vector according to claim 9, which is a plasmid containing a promoter derived from phage T7, preferably selected from pET11a-d、pET15b、pET19b、pET28a-c(+)、pET21a-d(+)、pET22b(+)、pET23a-d(+)、pEt25b(+)、pET44a-c(+)、pET46Ek/LIC, most preferably pET11a.
  11. 11. A prokaryotic expression system transformed with a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence as defined in claim 7 or 8 and/or a recombinant sequence comprising a part of a region comprising a recombinant DNA sequence as defined in claim 7 or 8.
  12. 12. The expression system of claim 11, which is an e.
  13. 13. A recombinant EJ17zipR hybrid protein, it consists of 1 ZIP domain, 56 elastin domains, 10 silk fibroin domains, 7 RGD domains 56 elastin domains, 10 silk fibroin domains, 7 RGD domains.
  14. 14. The recombinant EJ17zipR hybrid protein of claim 13, wherein the amino acid sequence contains 12-17% proline and/or 20-30% glycine.
  15. 15. The recombinant EJ17zipR hybrid protein of claim 13 or 14, wherein the protein has a molecular weight of 86kDa.
  16. 16. Recombinant EJ17zipR hybrid protein according to one of claims 13 to 15, having the amino acid sequence of seq#4.
  17. 17. Recombinant EJ17zipR hybrid protein according to one of claims 13 to 16, which constitutes a fragment of said recombinant protein.
  18. 18. Recombinant EJ17zipR hybrid protein according to one of claims 13 to 17, which is at least partially methacrylated.
  19. 19. A recombinant DNA sequence selected from the group consisting of: A recombinant DNA sequence encoding a recombinant EJ17zipR hybrid protein as defined in any one of claims 13 to 16, A recombinant DNA sequence comprising a region comprising a DNA sequence encoding a recombinant EJ17zipR hybrid protein as defined in any one of claims 13 to 16, and A recombinant DNA sequence which hybridizes to a DNA sequence encoding a recombinant EJ17zipR hybrid protein as defined in any one of claims 13 to 16.
  20. 20. The recombinant DNA sequence according to claim 19, having a sequence at least 80% identical, more preferably at least 90% identical, most preferably homologous to the recombinant DNA sequence of seq#2.

Description

Recombinant proteins, recombinant DNA sequences, vectors, eukaryotic and prokaryotic expression systems and uses thereof Technical Field The present invention relates to recombinant RE15mR and EJ17zipR hybrid proteins with the structures described in the specification, recombinant DNA sequences encoding the recombinant hybrid proteins, vectors containing the recombinant DNA sequences, and eukaryotic and prokaryotic expression systems transformed with the recombinant DNA sequences. The invention also relates to the use of the recombinant hybrid protein as a component of a bio-ink intended for bio-printing and as a component of a biological material. Furthermore, the invention relates to the use of said recombinant DNA sequences, vectors and eukaryotic or prokaryotic expression systems for the preparation of recombinant hybrid proteins. The invention can be used for biological printing, medicine and cell and tissue engineering. Background Bioprinting and biomaterials are currently under intense research and development. The technical solutions in these fields enable 3D printing of tissue structures and are ultimately applied in regenerative medicine. Thus, examples of natural and synthetic bio-inks and biomaterials are known in the art, such as biopolymers disclosed in U.S. patent No. 2022/0047706. However, there is still a need for materials used in bioinks and biomaterials that improve their impact on cell viability, proliferation, adhesion, or cytotoxicity (which may also be combined as components of a blend). Object of the Invention The object of the present invention is to propose a novel, alternative recombinant hybrid protein, which is particularly useful as a component of biological inks and biological materials. Such proteins should have versatility, safety and effectiveness, both in laboratory settings and ultimately in patients. The object of the present invention is also to overcome the technical problems found in the prior art, in particular by providing a protein which is comparable or superior to the proteins currently used in the prior art in at least one parameter, such as the effect on cell viability, proliferation, adhesion or cytotoxicity (which may also be combined as components of the blend), rheological parameters (modulus of the composite, dynamic viscosity), printability (printability, resolution, continuity and stability of the fiber), mechanical properties (compressive mechanical strength, young's modulus). Disclosure of Invention The first aspect of the invention relates to a recombinant RE15mR hybrid protein consisting of a sequence of 3 meromelanin domains, 1 k+ domain, 3 meromelanin domains, 1 MMP domain, 3 elastin domains, 1 k+ domain, 4 elastin domains, 1 k+ domain, 3 elastin domains, 1 MMP domain, 7 RGD domains in this order. Preferably, the amino acid sequence of the recombinant RE15mR hybrid protein according to the invention contains 12-15% proline and/or 32-38% glycine. Preferably, the recombinant RE15mR hybrid protein of the invention has a molecular weight of 26kDa. Preferably, the recombinant RE15mR hybrid protein of the invention has the amino acid sequence of seq#3. Preferably, the recombinant RE15mR hybrid protein of the invention is a fragment of a recombinant protein. Preferably, the recombinant RE15mR hybrid proteins of the invention are at least partially methacrylated. In a second aspect, the invention provides a recombinant DNA sequence selected from the group consisting of: a recombinant DNA sequence encoding a recombinant RE15mR hybrid protein as defined above, A recombinant DNA sequence comprising a region comprising a DNA sequence encoding a recombinant RE15mR hybrid protein as defined above, and A recombinant DNA sequence which hybridizes to a DNA sequence encoding a recombinant hybrid protein RE15mR as defined above. Preferably, the recombinant DNA sequence of the second aspect of the invention has a sequence which is at least 80% identical, more preferably at least 90% identical, most preferably homologous to the recombinant DNA sequence of seq#1. In a third aspect, the present invention provides a vector comprising a recombinant DNA sequence comprising a region of a recombinant DNA sequence according to the second aspect of the invention, and/or a recombinant sequence comprising a portion of a region of a recombinant DNA sequence according to the second aspect of the invention. Preferably, the vector according to the invention is a plasmid containing a promoter derived from phage T7, preferably selected from pET11a-d、pET15b、pET19b、pET28a-c(+)、pET21a-d(+)、pET22b(+)、pET23a-d(+)、pEt25b(+)、pET44a-c(+)、pET46Ek/LIC, most preferably pET11a. In a fourth aspect, the present invention provides a eukaryotic or prokaryotic expression system transformed with a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence according to the second aspect of the invention and/or a recombinant sequence comprising a part of a region comprising a reco