Preparation And Activity Identification Of A Novel Recombinant Human Fusion Collagen

Collagen is the most abundant protein in human body,accounting for about one third of the total protein in human body.It plays an important role in organ support,body protection,tissue damage repair and so on.It has been widely used in skin,tissue engineering,regenerative medicine,medical cosmetology and other aspects because of its good biocompatibility,promoting cell growth,adhesion and synergistic repair of trauma.In recent years,the global collagen industry has developed steadily and the domestic market demand has increased rapidly.At present,collagen is mainly extracted from animal skin and bone,but it may be contaminated by viruses,bacteria,toxins and other pollution,which poses the risk of the spread of animal borne diseases.At the same time,there are various extraction methods,such as acid,alkali and salt extraction,etc.,which have the disadvantages of low extraction rate and serious destruction of amino acid.The production of recombinant collagen by microbial fermentation can solve these problems well.Therefore,it is very important to establish a microbiological synthesis method for effective hydroxylation of recombinant collagen.Although recombinant human like collagen has been widely used,due to the lack of hydroxylase,it is difficult to achieve proline hydroxylation and high yield of macromolecular collagen in common exogenous expression systems such as Escherichia coli,Saccharomyces cerevisiae and Pichia pastoris at the same time.While proline hydroxylation is the most predominant post-translational modification of collagen molecules and is essential for stabilizing the triple-helix structure of collagen and participating in cellular receptor recognition.Therefore,it is crucial to establish a microbial fermentation method for efficiently hydroxylated recombinant human collagen.In this study,by co-expressing Bacillus anthracis proline hydroxylase and recombinant human fusion collagen gene in Escherichia coli,a recombinant human hydroxylated collagen containing multiple functional sites with higher thermal stability and better biocompatibility was obtained.Firstly,the recombinant human collagen was designed according to the cell receptor binding sequence in type I and type III collagen.Cell biological activity experiments showed that all recombinant proteins could well support and promote the growth of 3T3 cells,and the fusion protein with multiple motifs derived from both human typeⅠand III collagen exhibits better biocompatibility than single type I or type III recombinant collagen in the similar molecular weight.Among them,when the recombinant protein rh COL（Ⅰ+Ⅲ）₂ with a molecular weight of 120k Da was used as the matrix,the relative growth activity of the cells was up to81%.By comparing the hydroxylation ability of prolyl 4-hydroxylases from Bacillus anthracis（Ba P4H）,Arabidopsis thaliana（At P4H）,and Dactylosporangium sp.RH1（Da P4H）,it was found that Ba P4H had the best hydroxylation effect,and the hydroxylation rate was 44%.In order to improve the yield and proline hydroxylation rate of recombinant human collagen,the co-expression system was optimized.The hydroxylation rate of recombinant collagen in the two-plasmid co-expression system was significantly higher than that in the multiple-transcription unit single-plasmid co-expression system.The optimum shake flask culture conditions for the two-plasmid co-expression system were to use TB medium containing0.5 g/L arabinose at 37°C to cultivate to the mid-logarithmic growth phase,and 1 m M IPTG induction for 5 hours.Furthermore,a yield of hydroxylated collagen at 0.8 g/L was achieved by fed-batch fermentation in a 5 L fermenter.Compared with non-hydroxylated recombinant collagen,hydroxylated recombinant collagen showed significant improvements in thermal stability and biocompatibility.In conclusion,this study provided a recombinant human fusion collagen containing multiple functional sites,which was effectively hydroxylated by co-expressing Bacillus anthracis proline hydroxylase.The production of hydroxylated rh COL（I+III）₂ was further scaled up in a 5 L fermenter.It is of great significance to the application of recombinant collagen in the fields of food,medicine and cosmetics.