基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征
Expression,purification,and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9
何火聪 1林丽香 2李玲玲 3吴伦巧 4林海英 5潘剑茹5
作者信息
- 1. 福建医科大学附属肿瘤医院/福建省肿瘤医院放射生物学及肿瘤放射治疗学研究室,福建福州350014;福州大学化学学院,福建福州350108
- 2. 福州大学生物科学与工程学院,福建福州350108;雅博捷锐(厦门)医疗科技有限公司,福建厦门361028
- 3. 福州大学生物科学与工程学院,福建福州350108;福州捷赫生物科技有限公司,福建福州350108
- 4. 福州大学生物科学与工程学院,福建福州350108;深圳合民生物科技有限公司,广东深圳518116
- 5. 福州大学生物科学与工程学院,福建福州350108
- 折叠
摘要
融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤.然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果.为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合 MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9.该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞.全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达.GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47kDa,与理论值一致.纯化的融合蛋白的SOD活性和GST活性分别为2 954 U/mg和328 U/mgoGST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化.该融合蛋白在溶液中可被胶原酶Ⅳ部分水解.分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响.在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强.GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低.本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础.
Abstract
Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage.However,the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells,thus protecting tumor cells and consequently reducing the efficacy of radiotherapy.There are active matrix metalloproteinase(MMP)-2 or MMP-9 in most tumor cellular microenvironments.Therefore,a fusion protein containing an MMP-2/9 cleavable substrate peptide X,a cell-penetrating peptide R9,a glutathione S-transferase(GST),and a human Cu,Zn superoxide dismutase(SODI),was designed and named GST-SOD1-X-R9.In the tumor microenvironment,GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9,thereby achieving selected entering normal cells.The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1.The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained,and soluble expression of the fusion protein was achieved.GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography.The molecular weight of the fusion protein was approximately 47 kDa,consistent with the theoretical value.The SOD and GST activities were 2 954 U/mg and 328 U/mg,respectively.Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions.The fusion protein could be partially digested by collagenaseⅣ in solution.Subsequently,the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells.Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition.While under the 3D culture model,the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily.GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells,but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably.This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
关键词
抗氧化酶/MMP-2/9敏感性/可跨膜/大肠杆菌表达体系/纯化/稳定性Key words
antioxidant enzyme/MMP-2/9 sensitive/cell permeable/expression in Escherichia coli/purification/stability引用本文复制引用
基金项目
出版年
2022
NETL
NSTL
维普
万方数据