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Recombinant collagen as well as preparation method and application thereof

A technology for recombining collagen and collagen, applied in the field of collagen expression, can solve the problems of increasing the purification cost, increasing the complexity of the purification process, reducing the yield of full-length α1 chain, etc., and achieving the effect of increasing the yield

Active Publication Date: 2022-03-01
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such degradation not only reduces the yield of the full-length α1 chain produced by expression, but also requires two steps of double-affinity purification to obtain a high-purity single full-length α1 chain product because of its similar properties to the full-length α1 chain , which increases the complexity of the purification process and correspondingly increases the purification cost

Method used

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  • Recombinant collagen as well as preparation method and application thereof
  • Recombinant collagen as well as preparation method and application thereof
  • Recombinant collagen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Design and synthesis of amino acid sequences

[0073] The amino acid sequence of the human type I collagen α1 chain (denoted as α1(I)) refers to the sequence part 162–1218 (PRO_0000005720) of the Uniprot database P02452-1 (https: / / www.uniprot.org / uniprot / P02452), It is the amino acid sequence of the α1 chain of mature human type Ⅰ collagen, without the signal peptide, C-terminal propeptide, N-terminal propeptide and other parts that will be processed and shed in the α1(Ⅰ) precursor protein. Its sequence is shown in SEQ.ID.NO .1 shown.

[0074] SEQ.ID.NO.1:

[0075] QLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQ...

Embodiment 2

[0108] Embodiment 2. Construction of recombinant expression vector, strain screening

[0109] (1) Construction of recombinant expression vector

[0110] The synthesized gene fragments SEQ.ID.NO.8 and SEQ.ID.NO.10 were recombined into the pPIC9K empty vector (purchased from Thermo Fisher Scientific Corporation), so that the target fragments could be accurately inserted into the vector containing the secretion signal α- Two recombinant expression vector plasmids, pPIC9K-COL2A1M6 expressing α1(II)M6 and pPIC9K-COL1A1M1 expressing α1(Ⅰ)M1, were obtained in the reading frame of the secretion vector of the factor.

[0111] The pPIC9K-COL2A1M6 and pPIC9K-COL1A1M1 plasmids were transformed into competent Escherichia coli DH5α (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), positive clones were screened on LB resistance plates containing ampicillin, and recombinant plasmids were extracted for sequencing identification ( Completed by Sangon Bioengineering (Shanghai) Co., L...

Embodiment 3

[0119] Example 3. Induced expression and identification of recombinant collagen

[0120] Take the recombinant engineering bacteria expressing α1(I)M1 and α1(II)M6 obtained in Example 2 respectively, and simultaneously take the engineering strains of Pichia pastoris expressing full-length type I collagen α1 chain protein and expressing full-length II The Pichia pastoris engineering strain of type collagen α1 chain protein was used as a control, and the two control engineering strains were all the previous research results of the inventor's team. The expressed full-length collagen α1 chain also added a Strep-Tag II tag, carboxyl Add 6×His Tag tags at the end), which are respectively from the application number 201911135958.0 (name: yeast recombinant human type I collagen α1 chain protein, synthesis method and application, Pichia pastoris expressing the full-length α1(I) chain in the patent The engineering strains are preserved in the General Microbiology Center of China Microbio...

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Abstract

The invention relates to recombinant collagen as well as a preparation method and application thereof, in particular to a recombinantly expressed full-length collagen alpha1 chain as well as a preparation method and application thereof, and belongs to the technical field of collagen expression. When the human I-type collagen alpha1 chain (alpha1 (I) chain) variant (recorded as alpha1 (I) M1) and the human II-type collagen alpha1 chain (alpha1 (II) chain) variant (recorded as alpha1 (II) M6) are subjected to recombinant expression in pichia pastoris, the human I-type collagen alpha1 chain (alpha1 (I) chain) variant (recorded as alpha1 (I) M1) and the human II-type collagen alpha1 chain (alpha1 (II) chain) variant (recorded as alpha1 (II) M6) are subjected to recombinant expression in pichia pastoris; a main degradation product (main degradation band) which is basically the same as a full-length alpha 1 chain target product (target band) in proportion and appears during recombinant expression of a natural full-length alpha 1 (I) chain and a natural full-length alpha 1 (II) chain is eliminated, so that the yield of the target product is increased; compared with natural full-length alpha 1 (I) chain collagen and alpha 1 (II) chain collagen which are subjected to recombinant expression in pichia pastoris, the alpha 1 (I) chain collagen has similar physicochemical characteristics and biological activity, and has the value of being applied to the field of biomedical materials.

Description

technical field [0001] The invention relates to recombinant collagen, a preparation method and application thereof, in particular to a recombinantly expressed full-length collagen α1 chain, a preparation method and application thereof, and belongs to the technical field of collagen expression. Background technique [0002] Type Ⅰ and type Ⅱ collagen are typical fibroblast collagens in the human body, both of which are composed of three α-peptide chains, and each α-peptide chain contains an amino-terminal peptide region, a characteristic (G-X-Y)n triple repeat sequence region, The carboxy-terminal peptide region has three parts. [0003] Type I collagen is composed of two α1 chains and one α2 chain, and is the most abundant type of collagen in the human body. It exists in muscle, skin, arterial wall, and fibrocartilage. Type II collagen is composed of three α1 chains, mainly distributed in cartilage tissue, vitreous body, and cornea, accounting for more than 90% of the total...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/81C12N1/19C12P21/02C07K1/18C12N5/077A61L27/24A61L27/52C12R1/84
CPCC07K14/78C12N15/815C12N5/0656A61L27/24A61L27/52C12N2533/54Y02A50/30
Inventor 李佳佳王丽萍刘慧敏蒋雯雯钱晨明程鹏飞钱松
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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