Modified bacterial collagen-like proteins

一种胶原蛋白、蛋白的技术，应用在修饰的细菌胶原蛋白样蛋白领域，能够解决复杂、增加系统复杂性等问题

Active Publication Date: 2016-07-13
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the generation of human mimics, the yeast system is complicated by the need to introduce the gene for proline-4 hydroxylase to form the Hyp residue required for the stability of mammalian collagen
Typically, recombinant mammalian-encoded collagen is expressed in Pichia, which requires the addition of oxygen for maximum hydroxylation and methanol for induction, adding to the complexity of the system

Method used

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  • Modified bacterial collagen-like proteins
  • Modified bacterial collagen-like proteins
  • Modified bacterial collagen-like proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0200] Example 1: Bacterial collagen sequences with Tyr residues introduced at the C-terminus

[0201] The combined globular and collagen-like portion of the scl2.28 allele (Q8RLX7) encoding the Scl2.28 protein, but lacking The DNA sequence of the fragment of the C-terminal linked domain is recorded as GenBank: AY069936.1. Introduce His into this sequence at the N-terminus 6 tag, and the thrombin / trypsin cleavage sequence LVPRGSP (SEQ ID NO: 1) was inserted between the N-terminal globular domain (V) and the following (Gly-Xaa-Yaa) n Between triple helix / collagen-like (CL) domain sequences. The triplet sequence GKY is included at the C-terminus of the CL domain, followed by a stop codon, with NdeI and BamHI cloning sites. DNA for this design was synthesized commercially without any codon optimization. SEQ ID NO: 2 is the final DNA construct. SEQ ID NO: 3 is the translated protein sequence.

Embodiment 2

[0202] Example 2: Bacterial collagen sequences with Cys residues introduced at the N-terminus

[0203] The DNA sequence of collagen containing triple helical repeats from Candidatus Solibacterusitatus Ellin 6076 was obtained as entry ABJ82342 from data provided in the National Institutes of Health, Bethesda, MD 20894, USA. The DNA sequence of the V domain from Rhodopseudomonas palustris was obtained as YP_001993084 from data provided in the National Institutes of Health, Bethesda, MD 20894, USA database. The protein sequence was translated into a nominal DNA sequence and a composite gene was designed maintaining the correct coding frame with the Met initiation signal followed by the additional amino acid sequence Gly-Cys-Pro containing Cys residues followed by the S The CL domain of .usitatus, followed by the V domain from Rhodopseudomonas palustris, and finally followed by the C-terminal His 6 tags and stop codons. Terminal restriction sites outside the coding sequence were...

Embodiment 3

[0204] Example 3: Bacterial collagen sequences with both Cys and Tyr residues introduced at the C-terminus

[0205] The sequence of this example was prepared as described in Example 1, except that the sequence at the C-terminus of the protein was Gly-Tyr-Cys at the end of the CL domain, followed by a stop codon and a restriction site, The DNA of this sequence was commercially synthesized without any codon optimization. The sequence of this construct is shown in SEQ ID NO:6 and 7.

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Abstract

The present disclosure relates to recombinant or synthetic collagen-like proteins comprising at least one triple-helical domain and wherein the collagen-like protein is modified compared to a native bacterial collagen-like sequence.

Description

[0001] Cross-references to other applications [0002] This application claims priority to Australian Provisional Patent Application No. 2013903444, entitled 'Modification of bacterial collagen-like proteins', filed 9 September 2014, the entire content of which is incorporated by reference into this article. [0003] All publications, patents, patent applications, and other references cited herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually are incorporated by reference as indicated. technical field [0004] The present disclosure relates to recombinant or synthetic collagen-like proteins comprising at least one triple helical domain, and wherein the collagen-like proteins are modified compared to native bacterial collagen-like sequences. Background technique [0005] Collagen is the major structural protein in the extracellular matrix ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78A61K38/16A61K8/65C12P21/02C12N15/31
CPCA61K8/65C07K14/78A61K38/00A61Q19/08Y02A50/30A61K2800/10C07K14/195
Inventor J·A·M·拉姆尚J·A·沃克迈斯特V·斯托伊切夫Y·Y·彭
Owner EVONIK OPERATIONS GMBH
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