Preparation of type I collagen-like fiber and method for regulating and controlling the D-periodic of fiber thereof

Patent metadata

Field	Value
Publication number	US-11639377-B2
Application number	US-202017082525-A
Country	US
Kind code	B2
Filing date	Oct 28, 2020
Priority date	Apr 23, 2020
Publication date	May 2, 2023
Grant date	May 2, 2023

Abstract

The disclosure discloses a preparation of type I collagen-like fibers and a method for regulating and controlling the periodic length of fiber stripes thereof, belonging to the technical field of genetic engineering. The disclosure produces a three-segment chimeric collagen P-CL-P pattern by inserting a continuous Gly-Xaa-Yaa triplet collagen sequence in the middle based on the N- and C-terminal (GPP)n sequences. The self-assembly is driven by the interaction between the N- and C-terminal (GPP)n triple helixes to form banded fibers with periodic bright and dark stripes. According to the method of the disclosure, a fiber from a clean source, which can self-assemble to form periodic bright and dark stripes can be prepared, the structure of which is similar to type I collagen, the preparation process is simple, the collagen fiber with low cost can be produced on a large scale, and the method has broad application prospects in the field of biological materials.

First claim

What is claimed is:

1. A single-chain protein for expressing a type I-like collagen, wherein:
the single-chain protein consists of an amino acid sequence as set forth in:

V-domain has an amino acid sequence as set forth in SEQ ID NO:1;
(GPP)n is (glycine-proline-proline)n;
n=10;
the CL-domain has an amino acid sequence as set forth in any sequence of SEQ ID NOS: 2 to 6;
HIS is a 6×His tag;
the V-domain and (GPP) n are ligated by LVPRGSP (SEQ ID NO:33); and
the single-chain protein self-assembles with additional single-chain proteins to form the type I-like collagen fibers with D-periodic band structure comprising uniform spacing of bright and dark alternating stripes when viewed with electron microscope.

2. The single-chain protein according to claim 1 , wherein the bright and dark stripes have a length of p×1 nm, wherein p is an integer greater than 5.

3. A gene encoding the single-chain protein according to claim 1 .

4. A plasmid or a cell, wherein the plasmid comprises the gene according to claim 3 , and wherein the cell comprises the gene according to claim 3 .

5. The plasmid or the cell according to claim 4 , wherein the plasmid is a pColdIII series plasmid or a pET series plasmid.

6. The plasmid or the cell according to claim 4 , wherein the cell is an E. coli cell selected from the group consisting of: E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, and E. coli TOP10.

7. A method for preparing type I collagen-like fiber, comprising the following steps:
(1) culturing the cell of claim 4 under conditions that induce expression of the gene;
(2) adding a trypsin to a purified gene product of step (1) and incubating at 20° C. to 25° C. for at least 6 hours to obtain the single-chain protein of the type I-like collagen; and
(3) adding the single-chain protein of a type I-like collagen obtained in step (2) to a solution at a final concentration of 0.1 mmol/L to 1 mmol/L, and storing the solution at 2° C. to 37° C.

8. The method according to claim 7 , wherein the gene in step (1) has a nucleotide sequence as set forth in any one of SEQ ID NOS: 13 to 15, 17, and 18.

9. The method according to claim 7 , wherein the storing time in step (3) is 24 hours or more.

10. The method according to claim 7 , wherein in step (3), the collagen is added to the solution at a final concentration of 0.5 mmol/L, and storing the solution at 4° C. to 37° C. for at least 2 days.

Assignees

• Univ Jiangnan
• Rutgers Univ

Inventors

• Xu Fei
• Hu Jinyuan
• Nanda Vikas
• Shreiber David I
• Zhang Meng
• Gahlawat Sonal

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C07K2319/21

  containing a His-tag · CPC title