Purification and characterization of MAP30 by immunoaffinity chromatography

doi:10.3877/cma.j.issn.1674-0785.2023.02.015

Abstract

Abstract:

Objective

To establish an immunoaffinity chromatography method for rapid purification of MAP30 polypeptide (a momordica toxin).

Methods

New Zealand white rabbits (male) were immunized with the toxin MAP30 as an antigen to prepare antiserum. Polyclonal antibodies were purified by Mabselect affinity chromatography. Synthesized antibody-Sepharose 2B affinity chromatography medium using Sepharose 2B as a matrix coupled with polyclonal antibody was used to purify the momordica toxin MAP30.

Results

The MAP30 toxin purified with the self-made immunoaffinity medium showed a single color band under reducing conditions on SDS-PAGE, and the corresponding relative molecular weights were all 30 kDa. The total sugar content measured by the sulfate-anthrone method was 1.54%. PC12 cells were used as the test cell type, and the maximum inhibition rate of MAP30 (anti-tumor activity) was 90%.

Conclusion

The structure, properties, and activity characteristics of the purified target protein-MAP30 in this study are consistent with those reported in the literature, which suggests that the immunoaffinity chromatography method established in this study is feasible for the separation and purification of MAP30.

Key words: Immunoaffinity chromatography, MAP30, RIP, Polyclonal antibody

Zihao Qiang, Yiping Zhou, Di Yang, Yanli Wu, Yongjia Zhang, Xiang Fan, Gangrui Li, Yanfa Meng, Yao Meng. Purification and characterization of MAP30 by immunoaffinity chromatography[J]. Chinese Journal of Clinicians(Electronic Edition), 2023, 17(02): 195-201.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhlcyszz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-0785.2023.02.015

https://zhlcyszz.cma-cmc.com.cn/EN/Y2023/V17/I02/195

Figures/Tables 7

References 26

1	Akkouh O, Ng TB, Cheung RC, et al. Biological activities of ribosome-inactivating proteins and their possible applications as antimicrobial, anticancer, and anti-pest agents and in neuroscience research [J]. Appl Microbiol Biotechnol, 2015, 99(23): 9847-9863.
2	Zhu F, Zhou YK, Ji ZL, et al. The plant ribosome-inactivating proteins play important roles in defense against pathogens and insect pest attacks [J]. Front Plant Sci, 2018, 9: 146.
3	Stirpe F. Ribosome-inactivating proteins: from toxins to useful proteins [J]. Toxicon, 2013, 67: 12-16.
4	Endo Y, Mitsui K, Motizuki M, et al. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins [J]. J Biol Chem, 1987, 262(12): 5908-5912.
5	Mundy J, Leah R, Boston R, et al. Genes encoding ribosome-inactivating proteins [J]. Plant Mol Biol Rep, 1994, 12(2): S60-S62.
6	Stirpe F, Battelli M G. Ribosome-inactivating proteins: progress and problems [J]. Cell Mol Life Sci, 2006, 63(16): 1850-1866.
7	Jiang Y, Miao J, Wang D, et al. MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression [J]. Oncol Lett, 2018, 15(4): 5833-5840.
8	Puri M, Kaur I, Kanwar RK, et al. Ribosome inactivating proteins (RIPs) from Momordica charantia for anti viral therapy [J]. Curr Mol Med, 2009, 9(9): 1080-1094.
9	Li M, Chen Y, Liu Z, et al. (2009) Anti-tumor activity and immunological modification of ribosome-inactivating protein (RIP) from Momordica charantia by covalent attachment of polyethylene glycol [J]. Acta. Biochim. Biophys Sin (Shanghai), 41(9): 792-799.
10	Bian X, Shen F, Chen Y, Wang B, Deng M, et al. (2010) PEGylation of alphamomorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential [J]. Biotechnol Lett, 32(7): 883-890.
11	Fang EF, Zhang CZ, Wong JH, et al. The MAP30 protein from bitter gourd (Momordica charantia) seeds promotes apoptosis in liver cancer cells in vitro and in vivo [J]. Cancer Lett, 2012, 324(1): 66-74.
12	Chan DW, Yung MM, Chan YS, et al. MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis [J]. Pharmacol Res, 2020, 161: 105157.
13	孟尧, 孟延发, 王济源. 一种同时制备苦瓜α-MMC和MAP30的方法 [P]. 上海市: CN112760310A, 2021-05-07.
14	Meng Y, Lin S, Liu S, et al. A novel method for simultaneous production of two ribosome-inactivating proteins, α-MMC and MAP30, from Momordica charantia L [J]. PloS One, 2014, 9(7): e101998.
15	史清洪, 彭冠英, 孙舒, 等. 环氧氯丙烷活化琼脂糖凝胶过程强化及性能评价 [J]. 过程工程学报, 2007, (4): 743-746.
16	March SC, Parikh I, Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography [J]. Anal Biochem, 1974, 60: 149-152.
17	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970, 227(5259): 680-685.
18	陶芳, 张文明, 姚大年. 酸性PAGE鉴定杂交水稻品种纯度的研究 [J]. 种子, 2007, (3): 31-34+37.
19	Doerner KC, White BA. Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain [J]. Anal Biochem, 1990, 187: 147-150.
20	王玉琪, 巫光宏, 林先丰, 等. 多糖和糖蛋白聚丙烯酰胺凝胶电泳染色方法的改进 [J]. 植物生理学通讯, 2009, 45, (2): 169-172.
21	刘春泉, 李大婧, 刘荣. 蒽酮-硫酸法测定北冬虫夏草多糖含量 [J]. 江苏农业科学, 2006, (2): 122-124.
22	詹少德. 花生过敏原Ara h2免疫亲和层析分离方法的建立 [D]. 南昌: 南昌大学, 2016.
23	姚利利, 席俊, 陈慧彬,等. 免疫亲和层析柱纯化大豆球蛋白多克隆抗体的研究 [J]. 中国食品学报, 2021, 21, (6): 296-300.
24	Yao X, Li J, Deng N, et al. Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia) [J]. J Sep Sci, 2011, 34(21): 3092-3098.
25	Yin J, Zheng H, Lin H, et al. Boronic acid-functionalized agarose affinity chromatography for isolation of tropomyosin in fishes [J]. J Sci Food Agric, 2019, 99(14): 6490-6499.
26	Zhang X, Yan Z, Wang Y, et al. Immunoaffinity chromatography purification and ultrahigh performance liquid chromatography tandem mass spectrometry determination of tetrodotoxin in marine organisms [J]. J Agric Food Chem, 2015, 63(12): 3129-3134.

[1]	Wenbo Gao, Ming Li, Yunhao Zhang. Prognostic value and clinical significance of advanced lung cancer inflammation index in patients with triple negative breast cancer [J]. Chinese Journal of Breast Disease(Electronic Edition), 2023, 17(04): 210-217.
[2]	Xiaoyan Zhang, Dongqiong Xiao, Hu Gao, Lin Chen, Fajuan Tang, Xihong Li. The protective effect and mechanism of overexpression of transcription factor 12 on the cerebral cortex of rats with sepsis-associated encephalopathy [J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2023, 19(05): 540-549.
[3]	Xiaoman Zhang, Xiaoqiu Ma, Zhengju Xu, Chunyu Zhang, Caiting He. Effect of drug-resistant mutations in reverse transcriptase region of hepatitis B virus on the level of serum hepatitis B surface antigen [J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2023, 17(05): 324-332.
[4]	Yansong Li, Huimin Feng, Mingchao Liu, Zepeng Liu, Qiuxia Jiang. Expression and clinical significance of STIP1 in triple negative breast cancer [J]. Chinese Journal of Operative Procedures of General Surgery(Electronic Edition), 2024, 18(01): 52-56.
[5]	Huixin Xu, Bo Liu, Lijun Tang. Development of a prediction model for failed extracorporeal shock wave lithotripsy of proximal ureteral stones larger than 1 cm [J]. Chinese Journal of Endourology(Electronic Edition), 2023, 17(05): 506-511.
[6]	Jinke Hu, Wen Zhong. Management and outcome of ureteral calculi in pregnancy [J]. Chinese Journal of Endourology(Electronic Edition), 2023, 17(04): 377-381.
[7]	Bin Wang, Jing Wang, Zansheng Huang, Ping Wang, Chuangye Wang, Shuoxin Zhang, Heng Qin, Xiaorong Sun, Hongmei Wu, Jin Hu, Yu Yang, Mingzhou Zhang, Li Li, Zhi Xu. Application of lung ultrasound visualization bronchoalveolar lavage in the diagnosis of peripheral lung lesions [J]. Chinese Journal of Lung Diseases(Electronic Edition), 2023, 16(04): 466-470.
[8]	Fangfang Huang, Yamei Chen, Muhong Deng, Xinhua Shi, Shaomei Shang. Risk factors analysis and prediction model of PICC-associated venous thrombosis in lung cancer patients [J]. Chinese Journal of Lung Diseases(Electronic Edition), 2023, 16(03): 324-328.
[9]	Haiyan Chen, Huimin Zhou, Jiaqi Cui. Moderate severe acute pancreatitis following transurethral cystoscopic holmium laser lithotripsy: report of one case [J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2023, 12(04): 461-463.
[10]	Lili Zhao, Kuixiang Wang, Xiaochong Zhang, Zhiyuan Li. Diagnostic value of ESR to C-Reactive protein ratio in diagnosis of periprosthetic joint Infection [J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2023, 09(06): 351-355.
[11]	Meina Li, Yanli Song, Shanshan Yang, Jucai Li, Huili Luo, Jie Lyu. Application and effectiveness evaluation of the triple pre-rehabilitation strategy in the perioperative period of patients with degenerative scoliosis [J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2023, 09(06): 356-364.
[12]	Ze Zhang, Qi Wang, Wenqiang Yang, Li Zhang. Application of musculoskeletal ultrasound in the diagnosis and treatment of peripheral neuropathy [J]. Chinese Journal of Brain Diseases and Rehabilitation(Electronic Edition), 2023, 13(05): 257-263.
[13]	Xin Sun, Haibo Cheng, Weixing Shen. Curative effect mechanism of Xianlian Jiedu decoction in treatment of patients with stage Ⅲ colorectal cancer based on transcriptomics [J]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2023, 13(05): 277-283.
[14]	Qiang Ma, Jun Li, Lijuan Gou. Expression levels of miR-21-3p and RUNX3 in severe acute pancreatitis and their predictive value to the progression of the disease [J]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2023, 13(05): 337-341.
[15]	Zhilong Zhang. Theory and application of acupuncture method of regulating spleen and stomach [J]. Chinese Journal of Acupuncture and Moxibustion(Electronic Edition), 2023, 12(03): 89-90.

Please choose a citation manager

Content to export