Preparation of recombinant humanized type III collagen and its structural characterization and safety evaluation

Abstract

BACKGROUND: Recombinant collagen can avoid the risk of viral transmission associated with animal-derived collagen and has good water solubility and excellent biological properties. It holds broad application prospects in medical, cosmetic, and food fields. However, there is a lack of systematic reports on strain construction, production process, structural characterization, quality research, and safety evaluation. OBJECTIVE: To construct a high-yield strain of recombinant humanized type III collagen, establish fermentation and purification processes, and characterize and evaluate the safety of the purified product. METHODS: A recombinant humanized type III collagen-expressing Escherichia coli strain was constructed. High-density fermentation was used to achieve high expression of the target protein. The target protein — recombinant humanized type III collagen — was extracted using immobilized metal affinity chromatography and ion exchange chromatography. The impurity residue and structure of the recombinant humanized type III collagen were analyzed by quantitative PCR, ELISA, ultra high performance liquid chromatography-mass spectrometry, and differential scanning calorimetry. The safety of the recombinant humanized type III collagen was evaluated through intradermal reaction test, skin sensitization test, acute systemic toxicity test, cell proliferation and cell migration experiments. RESULTS AND CONCLUSION: The constructed high-yield strain achieved a yield of 10 g/L in a 5 L fermenter. The peptide coverage and molecular mass of the purified product were consistent with the designed sequence. The melting temperature of the purified product was 79.72 °C, far above body temperature. Residual exogenous DNA, E. coli proteins, and bacterial endotoxins met standard requirements. Intradermal reaction, skin sensitization, acute systemic toxicity, cell proliferation and migration tests indicated that the recombinant humanized type III collagen product has good safety.

1. Introduction

Collagen is the most abundant protein in mammals, accounting for about 30% of total protein and three-quarters of skin dry weight, and is the most abundant component in the extracellular matrix [1-2]. The collagen family comprises 28 members, each containing at least one triple-helix domain. Collagens are deposited in the extracellular matrix and mostly form supramolecular aggregates [1,3]. Collagen is mainly distributed in human skin, bone, eyes, teeth, tendons, cardiovascular system, and other sites, and is closely related to tissue formation and support, intercellular information transmission, and plays important roles in joint lubrication, wound healing, blood coagulation, and cell aging [4-5]. Due to its excellent biological properties, collagen is widely used in medical materials, cosmetics, and food industry [6-10].

Collagen is generally extracted from tissues such as cartilage, skin, and tendon, using acid, alkali, or enzymatic methods. The difficulty of collagen extraction technology lies in virus inactivation of animal-derived materials, improvement of product purity, and limited raw material supply [11-12]. With the advancement of bioengineering technology, the development of recombinant collagen has become one of the current research hotspots. The YY/T 1849-2022 "Recombinant Collagen" proposed by the National Medical Products Administration and under the jurisdiction of the National Institutes for Food and Drug Control [13], and YY/T 1888-2023 "Recombinant Humanized Collagen" under the jurisdiction of the Center for Medical Device Evaluation of the NMPA [14] have been successively released, accelerating the development and application of recombinant collagen products in China. How to increase the yield of recombinant collagen, reduce production costs, improve product stability, and ensure product safety [15-16] has always been the focus of research.

Currently, most industrialized recombinant collagens use Escherichia coli or Pichia pastoris as host strains. E. coli has high collagen yield but has the risk of endotoxin residue, while yeast induced expression requires methanol, posing safety hazards [16]. LIU Di et al. [17] expressed a partial collagen domain fragment of human type I collagen α1 chain in yeast GS115, and by fusing telopeptides, the thermal denaturation temperature of the collagen-like product was increased to 39.9 °C, higher than body temperature 37 °C, and it had good biocompatibility and high application potential. TENG Fei et al. [18] achieved a yield of 1.43 g/L in 5 L fermentation by fusing human type I collagen with SUMO and 6×His tags in E. coli. WANG et al. [19] achieved high-level secretory expression of recombinant humanized type III collagen α1 in Pichia pastoris through multi-level systematic optimization, reaching a yield of 10.3 g/L in a 5 L fermenter, and the recombinant humanized type III collagen α1 exhibited significant antioxidant capacity and good biological activity, laying a foundation for its application in biomedical materials.

Obtaining collagen through recombinant expression avoids the risk of pathogen transmission and makes the production process more flexible and controllable [19-24]. The expressed protein needs to be effectively purified and characterized.