ELP-SOD融合蛋白的纯化及其脂质体制备

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Abstract Objective： The ELP-SOD fusion protein was expressed by recombinant E.coli cells, and purified ELP-SOD enzyme with high activity was obtained by three rounds of inverse transition cycling (ITC). ELP-SOD liposomes with long halflife and enzyme activity were prepared by liposome encapsulation technology, which laid a foundation for the application of recombinant human SOD in medical, health care, cosmetics and other fields. Methods： The purity of ELP-SOD was evaluated by SDS-PAGE, Tanon3500 gel imaging system and Image studio software; the enzyme activity of SOD fusion protein was measured using an improved pyrogallol autooxidation method (325 nm method); by adjusting different pH values and temperature gradients, the pH and thermal stability of the fusion protein were assessed; adding Cu2+/Zn2+ increased ELP-SOD enzyme activity; in vivo half-life experiments was simulated using rat plasma; cytotoxicity was evaluated by incubating HUVEC and L929 cells with different concentrations of ELP-SOD in culture media; preparation of ELP-SOD liposomes by thin film hydration method and determination of their encapsulation efficiency; mouse skin in vitro was prepared and in vitro release experiments were conducted using TP-6 transdermal diffusion apparatus. By using Franz diffusion cell the transdermal efficiency of ELP-SOD liposomes was to be investigated in carrying ELP-SOD. Results： The highest expression level of ELPSOD fusion protein was 10 mg/L, with a purity of 978% and an enzyme specific activity of 4 894.5 U/mg; its stability was higher at pH 5.0-8.0 and temperatures below 60 ℃; the access of ELP had a certain extension effect on the half-life of SOD and has good biosafety; the encapsulation efficiency of ELP-SOD liposomes reaches over 80%, which could further prolong the half-life of ELP-SOD, and its transdermal efficiency was significantly higher than that of ELP-SOD. Conclusion： The purification process of ELP-SOD fusion protein was simplified through reversible thermal denaturation technology, and combined with lipid preparation technology, the biological half-life of the enzyme protein was extended and its bioavailability was improved.

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Received: 03 August 2023

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	Articles by authors
	ZHU Yuerong1
	CHEN Xingyu2
	FENG Jiaoyan2
	ZHOU Ben2
	FAN Yanrong2

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ZHU Yuerong1,CHEN Xingyu2,FENG Jiaoyan2等. Purification of ELP-SOD fusion protein and liposome encapsulation[J]. Journal of Jiangsu University(Medicine Edition), 2024, 24(01): 44-51.

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http://zzs.ujs.edu.cn/xbyxb/EN/ OR http://zzs.ujs.edu.cn/xbyxb/EN/Y2024/V24/I01/44

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