Recombinant collagen - collagen types and extraction methods

(I) Types of collagen
In vertebrates, collagen is the main component of specialized and non-specialized connective tissues, accounting for almost a quarter of the total protein in the human body, three-fourths of the dry weight of human skin, more than 90% of human tendon and corneal tissue, and 80% of the organic matter in the bone consists of collagen.

The collagen molecule consists of two non-helical regions at both ends and a triple helix region in the middle. The collagen triple helix (tertiary structure) has a coiled helix structure consisting of three parallel α polypeptide chains (secondary structure), which are intertwined with each other in the form of regular helixes to form a rope-like structure with a molecular weight of about 300,000 g/mol, a length of 280 nm and a diameter of 1.4 nm. Intramolecular hydrogen bonds between glycines in adjacent chains stabilize the triple helix. Hydroxyl groups of hydroxyproline residues also form hydrogen bonds and stabilize the triple helix. Part of the collagen triple helix structure is composed of three identical alpha chains and is called the homotrimeric type (type II/III collagen). The triple helix sequence consists of Gly-X-Y repeats, with X and Y usually being proline and 4-hydroxyproline, respectively.

Twenty-eight types have been identified to date, and collagen types can be broadly divided into fibrous (types I, II, III, V, XI, XXI, XXVII) and non-fibrous (types IV, VI, VII, VIII, IX, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XVII, XVII, XVIII, XIX, XVII, XVII, XVII, XVII, XVII, XVIII, XVII, XVII, XVIII, XIX, XVII, XVIII, XVIII, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII types).

Non-fibrillar collagens can be divided into reticular collagens, beaded filamentous collagens, anchoring fibrils, membrane proteins, and multiplexins.

Fibrillar collagen types I, II, and III account for 80% to 90% of the collagen in the human body. Type I collagen, as the most common collagen in the human body, is mainly found in the skin, tendons, and bones; type II is mainly found in cartilage; and type III is mainly found in the skin and the vascular system.4 The collagen types I, II, and III are the most common in the human body.

(ii) Collagen synthesis and production
1. Collagen biosynthesis
Collagen biosynthesis can be divided into 2 stages, intracellular and extracellular, as shown below: intracellular synthesis and extracellular maturation stage.

The genetic information of each peptide chain of collagen molecule is transcribed by messenger RNA (mRNA) to ribosome, where peptide chains with more than 1,000 amino acid residues are synthesized, and the peptide chains are transferred to endoplasmic reticulum (ER) for hydroxylation and glycosylation modification.

(1) Hydroxylation modification: In the endoplasmic reticulum, peptide chains are generated from proline and lysine residues catalyzed by prolyl hydroxylase and lysyl hydroxylase. Hydroxylation plays an important role in the firmness of the triple-stranded helix, and insufficiently hydroxylated peptide chains can not form a firm triple-stranded helix at body temperature, and thus cannot be It cannot be secreted to the outside of the cell.

(2) Glycosylation modification: In the endoplasmic reticulum, peptide chains are formed by galactosyltransferase and glucosyltransferase catalyzed by attaching sugar groups to 5-hydroxylysine residues, which is conducive to the orientation of the fiber. After hydroxylation and glycosylation, soluble collagen can form three-stranded helical pre-collagen and be secreted outside the cell.

Three-stranded helical pre-collagen secreted into the extracellular lysogen by the action of endonuclease, hydrolysis of N-terminal and C-terminal additional peptide chains, the formation of proto-collagen, proto-collagen molecules can be in the neutral pH conditions, through the intermolecular parts of the different charges of the attraction of each other and the automatic polymerization of collagen fibers, the polymerization of the instability of the need for the lysine oxidase (lysyl oxidase) catalyzed by the lysine into aldolyl lysine (aldolyl oxidase) catalyst. The polymerization is unstable and needs to be catalyzed by lysyl oxidase, which transforms lysine into aldol lysine (allysine). After ε-aldehyde lysine is first condensed with ε-lysine aldol on the α-peptide chain to form ε-aldehyde lysine aldol, and then reacted with histidine to form alcohol aldol histidine, which is then condensed with aldol amine to form the Schiff base structure with 5-hydroxylysine, which can make the four α-peptide chains covalently cross-linked. Crosslinking, so that the tension of collagen microfibers strengthened, toughness increased, solubility decreased, and ultimately the formation of insoluble collagen fibers.

2、Collagen production
Collagen's good biocompatibility is widely used in biomedicine, the current raw material sources of collagen are broadly divided into four, tissue extraction, or from in vitro growth of human or animal cells, or through recombinant expression or direct peptide synthesis to the production of collagen raw materials used in the application.

(1) Tissue extraction method:

Mammalian skin and tendon tissues (derived from pigs, cows and sheep) are the main sources of type I collagen, while type II collagen is mainly extracted from cartilage tissues of cows, pigs and chickens. Due to the impact of foot-and-mouth disease and mad cow disease, people have begun to use fish scales, fish bones and fish skin as raw materials for collagen preparation. About 30 % of the by-products of aquatic product processing are fish skin and bone, which have high collagen content and are safer than collagen from terrestrial animal sources. Collagen extraction from other collagen-rich processing by-products, including egg membranes, chicken skin, bullfrog skin and rabbit, has also been investigated.

The principle of collagen extraction is to separate collagen with different characteristics from raw materials by changing the external environment (temperature, salt concentration, pH). Depending on the raw material, collagen can be extracted by a variety of methods, mainly including hot water method, acid method, alkaline method, enzyme method and salt method. Incoming researchers usually use a series of composite methods to extract collagen in order to get a higher collagen extraction rate during the extraction process while ensuring that the collagen is structurally intact and has good applicability.

Limitations of the tissue extraction method:

(i) Collagen structure destruction. The extraction process may disrupt the structural integrity of collagen and result in the destruction of tryptophan, serine and tyrosine.

(ii)High inter-batch variability of collagen products. The variability in the age and strain of the tissue raw material source causes significant variability between batches of extracted collagen products.

(iii) Risk of disease transmission. When using bovine collagen, there may be a risk of transmission of zoonotic diseases such as mad cow disease and foot-and-mouth disease.

(2) Chemically synthesized collagen

Gómez-Guillén9 et al. generated protofibrils using triple-helix tendency self-assembly technology to mimic the structure and thermal behavior of natural collagen, controlling the stability and self-assembly length of collagen by regulating the amino acid composition, temperature, and solvent.Falk10 added Fe2+ to a solution of triple-helix collagen-related peptide (collagen-related peptide, CRP ) in solution triggered self-assembly into morphologically diverse protofibrils. Although the chemical synthesis of triple-helical collagen has solved the problems of immune rejection and viral potential, it still has the following limitations:

(i) short sequence length. At present, the length of collagen prepared by chemical synthesis is usually not more than 10 nm, and it is difficult to realize the preparation of full-length collagen.

(ii) High cost and low yield. The amino acid monomers used in chemical synthesis of collagen have complex protective groups, and such reagents are usually expensive, resulting in high synthesis costs and difficulty in realizing large-scale production.

(iii) Organic solvent residue. In the process of chemical synthesis of collagen, a large number of organic solvents such as N,N-dimethylformamide (DMF) and dichloromethane (DCM) are used, which can easily cause the residue of organic reagents and lead to biotoxicity.

(3) Recombinant collagen

Recombinant collagen refers to the use of genetic engineering technology to produce collagen, the characteristics of human collagen and the main functional domains to optimize the design of the gene sequence, and then through the selection of a variety of host cells, such as genetically modified mice, insects, genetically modified silkworms, genetically modified tobacco, Escherichia coli, yeast and so on to produce recombinant human collagen. The collagen produced by this technology has the advantages of high safety, stable batch, single component, high activity and no immune rejection.