Optimization of HSP65-MUC1 Purification in Pilot Scale and Identification of Methods to Detect Biological Function_West China Medical Journal

Authors：

 ZHANGCheng ^1,2 , ZHANGHai-long ¹ , SUXiao-qing ¹ , LUOYi ¹ , FANQi-hao ¹ , LUOQi-ling ¹ , XIELi ¹ ,  LIYuan-bo ³ , YANGLi ¹

1. State Key Laboratory of Biotherapy, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Chengdu Xinlibang Bio-pharmaceutical Co., Ltd, Chengdu High & New Technology Industries Development Zone(West District), Sichuan 611731, P. R. China;
3. Yangtze River Pharmaceutical Group Sichuan Hairong Pharmaceutical Co., Ltd, Chengdu, Sichuan 610017, P. R. China;

Corresponding author：

LIYuan-bo, Email: 13908208669@139.com

Keywords：

HSP65-MUC1; Pilot scale production; Biological activity; Method

DOI：

10.7507/1002-0179.20140566

Video：

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Objective To optimize HSP65-MUC1 fusion protein purification in pilot scale through protein purification techniques and identify the methods for biological activity detection. Methods E. coli expressing HSP65-MUC1 was obtained by fermentation, then homogenized to obtain the supernatant. To acquire high-purity, high-quality HSP65-MUC1, the supernatant was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column purification. The expression of CD86 on the surface of DC cells treated with HSP65-MUC1 was determined with flow cytometry. Results E. coli containing pET28a-HSP65-MUC1 recombinant plasmid can effectively express target protein. A total of 413.7 mg of HSP65-MUC1 was obtained after 10 g of fermented cells was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column, and the purity was nearly 96%. Compared with negative control (10.13%±0.89%), purified HSP65-MUC1 could significantly improve the expression of CD86 on the surface of DC cells (29.98%±1.02%). Conclusion The pilot scale production of purified HSP65-MUC1 has been effectively optimized, and the methods of its biological activity detection have been identified, which simultaneously provides the basis for clinical studies.

Citation： ZHANGCheng, ZHANGHai-long, SUXiao-qing, LUOYi, FANQi-hao, LUOQi-ling, XIELi, LIYuan-bo, YANGLi. Optimization of HSP65-MUC1 Purification in Pilot Scale and Identification of Methods to Detect Biological Function. West China Medical Journal, 2014, 29(10): 1868-1873. doi: 10.7507/1002-0179.20140566 Copy

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13.	Feng Y, Wan M, Xiang ZM, et al. Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli[J]. Protein Expr Purif, 2007, 53(2):390-395.
14.	曹昭. HSP-MUC1纯化工艺的研究[D]. 长春:吉林大学, 2007.
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1. Li DP, Li H, Zhang PY, et al. Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity[J]. Eur J Immunol, 2006, 36(5):1324-1336.
2. Somersan S, Larsson M, Fonteneau JF, et al. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells[J]. J Immunol, 2001, 167(9):4844-4852.
3. 向泽敏, 吴秀丽, 万敏, 等. 热休克蛋白65-MUC1抗原肽融合蛋白效力测定方法的建立[J]. 中国生物制品学杂志, 2005, 18(4):332-335.
4. Sengupta D, Norris PJ, Suscovich TJ, et al. Heat shock protein-mediated cross-presentation of exogenous HIV antigen on HLA class Ⅰ and class Ⅱ[J]. J Immunol, 2004, 173(3):1987-1993.
5. Yuan SF, Shi CH, Lv YG, et al. A novel bacillus Calmette-Guerin-based breast cancer vaccine that coexpresses multiple tandem repeats of MUC1 and CD80 breaks the immune tolerance and inhibits MUC1-Positive breast cancer growth[J]. Cancer Biother Radiopharma, 2009, 24(5):607-613.
6. Joshi MD, Ahmad R, Yin L, et al. MUC1 oncoprotein is a druggable target in human prostate cancer cells[J]. Mol Cancer Ther, 2009, 8(11):3056-3065.
7. Qamra R, Mande SC. Crystal structure of the 65-kilodalton heat shock protein, chaperonin 60.2, of Mycobacterium tuberculosis[J]. J Bacteriol, 2004, 186(23):8105-8113.
8. Cao Z, Feng Y, Wei HF, et al. Purification of clinical-grade recombinant HSP65-MUC1 fusion protein[J]. Biotechnol Appl Biochem, 2010, 57(1):9-15.
9. Loveland BE, Zhao A, White S, et al. Mannan-MUC1-pulsed dendritic cell immunotherapy:a phaseⅠ trial in patients with adenocarcinoma[J]. Clin Cancer Res, 2006, 12(3 Pt 1):869-877.
10. Yang M, Yan YY, Fang ML, et al. MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1(+) tumor immunity in mice[J]. Int Immunopharmacol, 2012, 13(4):408-416.
11. 郑玉玲, 江华, 王希良, 等. HSP65-MUC1 VNTR2融合蛋白的表达、纯化及其抗肌瘤作用[J]. 细胞与分子免疫学杂志, 2007, 23(11):1014-1016.
12. Huo Y, Li B, Zhang YQ, et al. Pre-clinical safety evaluation of heat shock protein 65-MUC1 peptide fusion protein[J]. Regul Toxicol Pharmacol, 2007, 49(1):63-74.
13. Feng Y, Wan M, Xiang ZM, et al. Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli[J]. Protein Expr Purif, 2007, 53(2):390-395.
14. 曹昭. HSP-MUC1纯化工艺的研究[D]. 长春:吉林大学, 2007.
15. Thole JE, Keulen WJ, Kolk AH, et al. Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in Escherichia coli K-12[J]. Infect Immun, 1987, 55(6):1466-1475.
16. Portaro FC, Ma HS, De AL, et al. The Mycobacterium leprae hsp65 displays proteolytic activity:mutagenesis studies indicate that the M.leprae hsp65 proteolytic activity is catalytically related to the HslVU protease[J]. Biochemistry, 2002, 41(23):7400-7406.
17. Brossart P, Schnider A, Dill P, et al. The epithelial tumor antigen MUC1 is expressed in hematological malignancies and is recognized by MUC1-specific cytotoxic T-lymphocytes[J]. Cancer Res, 2001, 61(18):6846-6850.

West China Medical Journal

Optimization of HSP65-MUC1 Purification in Pilot Scale and Identification of Methods to Detect Biological Function

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