Expression and functional identification of WT-/GOF-ADAMTS13 proteins

doi:10.3969/j.issn.2095-4344.0604

Abstract

Abstract:

BACKGROUND: ADAMTS13 cleaves Von Willebrand factor (VWF) to regulate its size, thereby preventing aberrant platelet aggregation and thrombus. Eukaryotic cells are usually selected to express recombinant ADAMTS13. However, it still lacks a clear and efficient way for its expression and purification.
OBJECTIVE: To find a suitable host cell and get stable cell lines to express WT-ADAMTS13 (wild-type) and GOF-ADAMTS13 (gain of function), so as to establish the purification method for high-purity ADAMTS13 proteins, thus studying their biological functions.
METHODS: GOF-ADAMTS13 recombinant plasmid was obtained by site mutation kit with the WT-ADAMTS13 recombinant plasmid as a template. The expression effects of CHO-S, CHO-K1, and 293T cells on ADAMTS13 were compared to select appropriate host cells. The proteins were purified by Ni affinity chromatography and gel filtration. The purity and specificity of WT-ADAMTS13 and GOF-ADAMTS13 were identified by 7.5% SDS-PAGE and western blot assay. The interaction between WT-/GOF-ADAMTS13 and VWF-A2 was detected by atomic force microscopy to validate the activity of the two proteins.
RESULTS AND CONCLUSION: Compared with CHO-S and CHO-K1 cell lines, 293T cell line was more suitable as expression host for WT-/GOF-ADAMTS13. After Ni column affinity chromatography and gel filtration purification, the concentration of purified WT-/GOF-ADAMTS13 was 103.7 and 149.7 mg/L, respectively with the purity of above 90%. Atomic force microscopy results showed that the adhesion frequencies of WT-/GOF-ADAMTS13 and VWF-A2 were 11.37% and 14.70%, respectively. These results suggest higher affinity of GOF-ADAMTS13 binding to VWF-A2 is consistent with recent ADAMTS13 conformational studies.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Metalloproteases, Microscopy, Atomic Force, Tissue Engineering

CLC Number:

R446

Yu Shanshan, Lin Jiangguo, Fang Ying . Expression and functional identification of WT-/GOF-ADAMTS13 proteins[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(3): 441-446.

Figures/Tables 2

鉴于胎牛血清在培养过程中无法除去，选择CHO-K1细胞和293T细胞含有血清的样品进行Ni柱亲和层析，结果显示CHO-K1细胞和293T细胞在100 mmol/L咪唑洗脱液处见图2A中第8，9号泳道和图2B中第4，5号泳道均有目标条带(Mr约190 000)和血清杂条带。经ImageJ分析，CHO-K1细胞中目的条带比例占比约20%，而293T细胞中占约50%。因此，ADAMTS13更易从293T细胞上清液中通过Ni柱亲和层析纯化出来，故选择293T细胞作为宿主细胞表达野生型与功能增强型ADAMTS13蛋白。 2.3 分子筛二次纯化上述纯化结果显示在Ni柱亲和层析后仍有约50%杂蛋白，且杂蛋白主要集中在Mr=70 000处。为了得到更高纯度的ADAMTS13，采用分子筛进行二次纯化。将亲和层析后收集到的100 mmol/L咪唑洗脱液透析后经过分子筛纯化，结果显示在Mr=190 000附近出现单一条带见图3。经ImageJ分析，目的蛋白ADAMTS13的条带占比在90%以上。因此，亲和层析结合分子筛纯化，可得到纯度大于90%的ADAMTS13。 2.4 野生型与功能增强型ADAMTS13蛋白鉴定和定量分析对纯化后的野生型与功能增强型ADAMTS13蛋白进行7.5%SDS-PAGE电泳，同时进行Western-blot鉴定，检测蛋白的大小和His标签的准确性。野生型与功能增强型ADAMTS13相对分子质量为190 000，SDS-PAGE电泳和Western-blot显示在Mr=190 000均有准确表达见图4。BCA蛋白定量得到野生型与功能增强型ADAMTS13蛋白的质量浓度分别为103.733，149.716 mg/L。 2.5 野生型与功能增强型ADAMTS13蛋白功能鉴定通过原子力显微镜测量野生型与功能增强型ADAMTS13与血管性血友病因子A2的相互作用，以此鉴定纯化蛋白的生物学功能。黏附实验示意图如图5A，2%BSA处理的培养皿与血管性血友病因子A2功能化探针的黏附频率分别为(3.00±2.38)%，(1.64±1.45)%见图5B，说明2%BSA能有效阻断探针与底板间的非特异性黏附。功能增强型ADAMTA13和野生型ADAMTS13与血管性血友病因子A2间的黏附频率分别为(14.70±5.27)%，(11.37±4.71)%，均显著高于对照组(P < 0.001)，说明野生型与功能增强型ADAMTS13具有生物学功能，都可与血管性血友病因子A2的发生结合，且功能增强型ADAMTA13与血管性血友病因子A2的结合能力要大于野生型ADAMTA13(P <0.05)，推测与ADAMTS13构象有关。"

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