Expression And Purification Of Recombinant Human Collagen-like And Its Application In Cosmetics

Objectives:Recombinant human collagen-like protein was designed and expressed in E.coli.A maximum yield was obtained by optimized the fermentation and purification process.The biologic activity of human collagen-like protein was determined and stabilization for storage was optimized.Following this,safety and risk assessment of human collagen-like were conducted for further use in vivo.Methods:The gene encoding recombinant human collagen-like was cloned into expression vector pET-3c,recombinant plasmid was constructed and named pET3c-hlcollagen,then recombinant plasmid was transformed into E.coli BL21(DE3),screened by ampicillin and induced by IPTG,the best expression strain was selected.Fermentation parameters were optimized and recombinant human collagen-like protein was purified through affinity chromatography NI Sepharose 6 Fast Flow combinated with gel filtration Sephadex G-25.Protectant formula are optimized by parallel contrast experiments to investigate the stability of recombinant protein concentrate and elite fluid preparations.Mouse fibroblast NIH / 3T3 cells were used for the cell adhesion assay.The safety of recombinant collagen-like protein was conducted through the acute dermal toxicity test,acute eye irritation test and skin allergy test.Results:1?DNA sequencing results showed that recombinant plasmid pET3c-hlcollagen matched exactly 100 % with the desired sequence.4 h after induction with 1 mM IPTG,the expression level of recombinant human collagen-like protein,molecular weight about 26 kDa,was 26 % of total protein in the soluble supernatant.Purified through affinity chromatography NI Sepharose 6Fast Flow combinated with gel filtration Sephadex G-25,the final yield of recombinant protein was approximate 500 mg/L fermention medium with the purity of up to 93.5 %.2?The results of adhesion activity experiments using NIH / 3T3 cells showed that recombinant human collagen-like protein had high cell adhesion characters,but showed no cell proliferation activity.3?Protectant formula screening results showed that recombinant human collagen-like protein reduced protein aggregation by adding 0.5 % alkyl glucoside.4?The selected protective agent is applied to the formulation of the essence,and a liquid essence product is developed.5?Given a maximum dose of 2 g /kg s.c.in mice,no death or organ toxicity happened in acute dermal toxicity test;no notable adverse events occurred on skin when received a dose of 10ug/cm2 on acute skin allergy test.These results suggested that the recombinant human collagen-like protein could be safe in animal experiments.Conclusion:Recombinant human collagen-like protein was efficiently expressed in E.coli.with a yield of 500 mg/L fermention medium by optimizing the fermentation and purification conditions.Recombinant human collagen-like protein had high cell adhesion character in vitro adhesion activity experiments using NIH / 3T3 cells.Alkyl glucoside was selected as optimal protective agent to avoid recombinant protein aggregation;In vivo animal experiments showed that the recombinant human collagen-like protein was safe without acute dermal toxicity,skin irritation and allergy etc.adverse effects.Altogether this study provided a stable recombinant human collagen-like protein essence by adding some additives for further use in market.