Yu Jianjun,Zhao Hua

Zhejiang Meishangjie Biotech Co.,Ltd.,China

Abstract By intercepting the most effective fragment of human collagen’s gene,recombinant humanlike collagen is produced by genetic engineering technology and biotechnology.The collagen has good biocompatibility and can promote cell formation.Recombinant human-like collagen has great potential application value in medical biological materials,cosmetics and foods.In recent years,many specialized enterprises which are engaged in human-like collagen raw materials production have emerged.In this paper,a brief summary of the current recombinant human-like collagen raw material’s production and its latest research and development have been made.

Key words recombinant human-like collagen;fibronectin;genetic recombination;biological fermentation

Collagen is a kind of biopolymer and it is the main component of animal’s hoof tissue.It is also the most abundant and most widely distributed functional protein in mammals,accounting for 25%～30% of total protein[1-3].Collagen is a kind of protein in the family of proteins.At present,more than 30 kinds of collagen gene codes have been deciphered,by which more than 16 kinds of collagen molecules can be synthesized.According to the different structure and function of collagen,it can be divided into fibrous collagen,basement membrane collagen,anchored collagen and so on.According to its different distribution in organism,collagen can be divided into interstitial collagen,basal collagen and peripheral collagen.Among them,interstitial collagen is major part of collagen in the whole organism,including type I,II and III collagen.Type I collagen mainly distributes in skin,tendon and other tissues,and it is the most widely used in medicine[4-6].Collagen consists of a special triple helix chain,and such a chain segment is called procollagen.Each collagen is made up of three α-helical peptide chains,each containing about 1,000 amino acid monomers.The most prominent feature of the chain is the periodic arrangement of amino acids (Gly-X-Y) n.In addition to providing structural support to the human body through periodic three-dimensional helical arrangements,collagen also plays an extremely important role in the formation of tissues and organs.

Recombinant human-like collagen is a protein with collagen like properties obtained by recombinant expression based on the optimization of the characteristics of human collagen or the introduction of special functional amino acid sequences.Compared with the traditional extracted collagen,it mainly has the advantages of processability,no virus hidden danger,good water solubility,stable production process and low rejection reaction[7].

When it comes to the tissue repair effect of collagen,fibronectin is needed to be mentioned.Fibronectin (FN) is a high molecular weight glycoprotein in extracellular matrix.It mainly exists in three forms:plasma fibronectin produced by hepatocytes or endothelial cells,cellular fibronectin secretory synthesized by fibroblasts and early mesenchymal cells,and fetal fibronectin in placenta and amniotic membrane.Fibronectin is widely involved in a variety of physiological activities of cells,including cell migration,adhesion,proliferation,hemostasis and tissue repair.In addition,fibronectin can also mobilize the mononuclear phagocyte system to remove harmful substances from damaged tissues,which is conducive to the rapid repair of wounds and has the effect of growth factor.As the basic material of cell culture,fibronectin can effectively improve the adhesion rate and confluence rate of cells,effectively shorten the time required for cell confluence,maintain the good morphological structure of cells and enhance the metabolic rate of cells.Most importantly,fibronectin can also significantly improve the synthesis speed of DNA,RNA and protein.By coating fibronectin on the microsphere carrier and using it as the medium for mass production of cells can save space and raw materials.It has become the basic material to prepare new drugs by large-scale cell culture technology[8].

1 Significance of the development of recombinant human-like collagen products

In recent years,collagen has been widely used in food,cosmetics,biomedical materials and other fields.Traditional collagen is mainly extracted from animal connective tissues.However,the collagen obtained from animal tissues is risky.First,it may have animal derived viruses.Second,the product quality may unstable due to the differences between animal individuals.Third,many people have allergies to animal derived proteins.These risks seriously limit the application and development of new collagen products[9].To a certain extent,this also reflects the necessity and urgency of the development of recombinant human-like collagen products.Especially in recent years,related research results have further confirmed that recombinant human-like collagen has made exciting progress in bone repair[10-15],skin wound repair[16-20],tendon repair[21-23],drug delivery[24-27],beauty[28,29]and other fields.

2 The latest research and development progress of human-like collagen products

2.1 Recombinant human-like collagen

Fan Daidi research group of Northwest University successfully used the genetically engineered strain E.coli BL 21 to ferment,isolate and purify human-like collagen.By using this technology,Fan’s group has successfully produced Type I recombinant human-like collagen in large quantities in cooperation with Xi’an Juzi Biotechnology Co.Ltd.Type I human-like collagen has lots of advantages,such as better water solubility,less immune rejection,better stability,better colloidality and absorptivity[30].Xi’an Juzi Biotechnology Co.Ltd.has recently launched a series of cosmetics based on type I human-like collagen.At the same time,based on the good skin repair function of type I collagen,Xi’an Juzi Biotechnology Co.Ltd.has also developed a variety of medical and beauty products,which are mainly aimed at sensitive skin care,acne repair and recovery after laser surgery and so on.

By cooperation with Fudan University-Jinbo functional protein research center and Sichuan University-Jinbo functional protein joint laboratory,the Shaanxi provincial key Laboratory (Functional protein Innovation Center) under Shanxi Jinbo Biomedical Co.Ltd.has developed the preparation process of type III recombinant human-like collagen.At present,this process is the only type III humanlike collagen process which has been successfully used in large-scale production.The preparation method is mainly to select the key fragment region 483aa-512aa of human’s type III collagen CO3A1 gene for repeated splicing and recombination.This type III human-like collagen has strong water solubility and high biological activity.The amino acid sequence of the basic repeat unit of Jinbo’s type III human-like collagen is 100% the same as that of the active functional region of human type III collagen,showing a 164.88 ° flexible bending structure,which can promote the close combination of the protein with human cells.The safety of type III human-like collagen developed by Jinbo Biomedical Co.Ltd.has passed the biological evaluation of the Institute of Food and Drug Control;Meanwhile,the company’s type III human-like collagen implant products have been tested by Sichuan Medical Device Biomaterials and Products Inspection Center and it has been confirmed to meet the requirements of class II medical device implantation[31].Jinbo biomedical Co.Ltd.has successfully developed a variety of type III human-like collagen functional surgical dressing products and medicinal type III human-like collagen solution products.In the field of skin care products,Jinbo Biomedical Co.Ltd.has also developed a series of products,such as facial mask,scalp repair.

Up to now,there are two kinds of collagen extraction technology,one is animal collagen extraction,the other is recombinant expression of collagen,and the latter is commonly divided into E.coli fermentation expression and yeast fermentation expression.It has to be mentioned here:because the animal source and production environment are not reliable,there are hidden dangers of viruses and allergens in the purification process of collagen,which also limits the further development of the potential use of animal derived collagen.However,there are still hidden dangers of biosafety and cell endotoxin in the fermentation and expression of E.coli.Moreover,collagen gene recombination has no modification activity and poor genetic stability.

In this regard,based on the sequence characteristics of collagen domain of human type III collagen,with the purpose of improving the water solubility and expression of collagen and by using protein engineering,molecular biology and gene recombination technology,the Yang Shulin research group of Nanjing University of Science and Technology designed and synthesized a gene monomer encoding with high hydrophilicity and repetitive Gly-X-Y tripeptide.The protein expression vector pPIc9KG6 containing six monomers was constructed in the same direction.The vector pPIc9KG6 was electrotransformed into Pichia pastoris,and the high copy recombinant yeast genetic engineering strain was screened by G418.At last,the expression of recombinant type III humanlike collagen is induced by methanol[32].Professor Yang has worked closely with Nanjing Yitongjin Biotechnology Co.Ltd.to develop various kinds of human-like collagen products,including type III recombinant human-like collagen mask paste,type III recombinant human-like collagen soft skin spray,robot nano sprayer.

By cooperating with Japanese professional beauty companies,Fulong group’s wholly-owned subsidiary-Chuang Ming medical devices Co.Ltd.has achieved the production capacity of the whole collagen industry chain,which is from the recombinant type III human-like collagen expression technology research and development,biological fermentation purification raw material production,to mask film cloth,final package design and production.The company has also launched a variety of medical beauty products,including yeast recombinant collagen gel dressing,yeast recombinant collagen oral mucosa repair solution,yeast recombinant collagen scar gel and so on.

2.2 Recombinant fibronectin human-like collagen

Wuxi Meishangjie Biotechnology Co.Ltd.cooperated with Swiss professional research institutions to creatively intercept the most effective fragment (COL1A1 part,intercepting the 498-788 gene sequence of type I collagen) from the human collagen genome.By fusing and expressing the COL1A1 part with amino acid sequence fragment of fibronectin (promoting adhesion growth),so as to promote cell adhesion and the expression of extracellular collagen.Recombinant fibronectin collagen can promote the increase of extracellular collagen,which plays a good role in moisturizing,repairing and wrinkle removing.While retaining the function of type I collagen,the molecular weight of recombinant fibronectin collagen is 32 KD (the average molecular weight of type I collagen is 300 kd),which is one tenth of type I collagen,and its molecular size is about 20～30 nm,which is more conducive to skin penetration and absorption.The reason why the company specially introduced the gene fragment of fibronectin is that fibronectin is an important component to promote wound healing.This component can quickly promote the cell adsorption,diffusion,replication and migration at micro level,so as to promote the repair of wound at the macro level,reduce or even avoid the formation of scar,enhance the tear resistance of wound and improve the healing speed of wound.Studies have proved that fibronectin can promote the rapid recovery of stubborn sores such as wounds,burns,scalds,postoperative wound nonunion,bedsore,sugar rotten feet,old sodden leg and so on.The fibronectin project to overcome chronic difficult healing wounds,jointly developed by Wang Deliang,Sheng Zhiyong and Fu Xiaobing,won the first prize of the achievement exhibition of the national innovation fund of China.Wuxi Meishangjie Biotechnology Co.,Ltd.innovatively introduced the key fragment of fibronectin,which is based on these research results.At present,Wuxi Meishangjie Biotechnology Co.,Ltd.has completed the R＆D and production of recombinant fibronectin human collagen(type I) expressed by yeast.They have also developed“MAIYL Meiyan QingJi” series cosmetics,which include skin care series,anti-aging makeup.

3 Future prospect of recombinant human-like collagen industry

Perhaps,some people will question that hyaluronic acid is also an important part of human skin,and the research and development of hyaluronic acid is quite mature.Based on this situation,is it necessary to further develop human like collagen products? Is there any suspicion of superfluous?However,in fact,although collagen and hyaluronic acid are both important components of human skin,they play completely different roles.If we use them to compare the raw materials for building houses,hyaluronic acid is equivalent to concrete,while collagen is equivalent to steel structure skeleton.It can be seen that both are indispensable for the repair of human skin.It is even necessary to supplement collagen to build a good skeleton,and then hyaluronic acid to achieve a better repair effect.It is for this reason that more and more enterprises are studying recombinant human like collagen products.

The reason why this paper focuses on the above enterprises is that these enterprises have their own collagen R＆D and production team,and they can also successfully put their research findings into mass production.As the application effect of recombinant human like collagen is affirmed by more and more people,there will be more and more related homogeneous products,and the market competition of such products will be more and more intense.If you want to occupy a place in this field,you need to be unique in the field of raw materials and product development,and the only way out is to develop unique and differentiated good products.