毕赤酵母工程菌表达乙肝表面抗原结合蛋白(SBP)的发酵纯化工艺研究

doi:10.13523/j.cb.20141209

中国生物工程杂志

2014, Vol. 34

Issue (12): 59-68 DOI: 10.13523/j.cb.20141209

技术与方法

毕赤酵母工程菌表达乙肝表面抗原结合蛋白(SBP)的发酵纯化工艺研究

董越, 苏彩霞, 杨艳, 朱乃硕

复旦大学生命科学学院及生物医学研究院遗传工程国家重点实验室上海 200433

The Study on Fermentation and Purification Process of HBsAg Binding Protein(SBP) in Pichia pastoris

DONG Yue, SU Cai-xia, YANG Yan, ZHU Nai-shuo

State Key Lab of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai 200433, China

全文: PDF(1674 KB) HTML

摘要：

乙肝表面抗原结合蛋白(HBsAg binding protein,SBP)是以HBsAg为探针,通过人肝cDNA噬菌体表达库筛选的一种人源蛋白。SBP可特异性结合乙肝表面抗原(Hepatitis B virus surface antigen, HBsAg),增强乙肝疫苗的免疫效果,是一种潜在快速高效的免疫佐剂。成功构建了分泌型高表达SBP的毕赤酵母工程菌。对该菌株进行了放大规模发酵表达,并对纯化工艺进行了研究。在发酵实时检测过程中,自诱导剂甲醇加入开始,SBP蛋白的分泌表达量随时间推移而逐渐增加,发现于38h达到最佳水平且此时杂蛋白含量最少,是放罐收集菌液的最佳时间。18L发酵液在低温离心除菌体和沉淀物后可得到15L的上清液,再利用截留量为5kDa的超滤膜包将上清液浓缩至2L,将浓缩后的上清液依次通过S200分子筛柱和TDEAE阴离子交换柱进行分离纯化,可得到300ml浓度为1.125mg/ml、纯度达98%的目标蛋白液,发酵液得率为22.5mg/L;最后将蛋白液定量分装冻干低温保存。所获得的放大规模SBP发酵诱导表达条件和SBP蛋白的分离纯化工艺,为SBP蛋白大规模生产奠定了坚实的基础。

关键词： 乙肝表面抗原结合蛋白; 毕赤酵母; 发酵; 纯化工艺

Abstract:

The HBsAg binding protein (SBP) was screened out from human liver cDNA phage expression library with HBsAg as a probe. SBP has the ability to enhance the immune response of Hepatitis B vaccine. It is proved to be a potential efficient immune adjuvant. The secretory SBP-expressing Pichia pastoris strains with high protein expression quantity has successfully constructed, then was fermented in the 18L fermentor. The results showed that SBP could achieve the maximum expression after 38h in methanol induction according to real time detecting; meanwhile hybrid proteins were the least so that it's the most appropriate time to collect bacteria liquid.15L supernatant could be obtained at low temperature centrifugation. Through the ultrafiltration membrane package whose interception was 5kDa, the supernatant was concentrated to about 2L. Then the concentrated fermentation supernatant was passed through sephadex S200 and the TDEAE column respectively. The attained 300ml SBP protein was with a purity of 98% and 1.125mg/ml as concentration. The SBP yield from fermented liquid was about 22.5mg/L.Finally, the lyophilized proteins were preserved at low temperature. The work on the fermentation process optimized the time, identified the purification process and lay a solid foundation for the mass production of SBP.

Key words: SBP Pichia pastoris Fermentation Purification process

收稿日期: 2014-09-02 出版日期: 2014-12-25

ZTFLH:

Q819

基金资助:

国家自然科学基金(5N136746,30901315,30970144)、国家"863"计划重大课题(2011AA02A114)、上海市科委科技支撑项目(13431900602)、国家新药创制重大专项(2009ZX0913710)、国家传染病重大专项(2012ZX10002006002003)资助项目

通讯作者: 朱乃硕 E-mail: nzhu@fudan.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

董越, 苏彩霞, 杨艳, 朱乃硕. 毕赤酵母工程菌表达乙肝表面抗原结合蛋白(SBP)的发酵纯化工艺研究[J]. 中国生物工程杂志, 2014, 34(12): 59-68.

DONG Yue, SU Cai-xia, YANG Yan, ZHU Nai-shuo. The Study on Fermentation and Purification Process of HBsAg Binding Protein(SBP) in Pichia pastoris. China Biotechnology, 2014, 34(12): 59-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141209 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I12/59

[1] 陈媛媛,朱乃硕.一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究.中国生物化学与分子生物学报,2005,21(1): 53-60. Chen Y Y, Zhu N S. Screening, expression and characterization of a novel protein binding to Hepatitis B surface antigen. Chin J Biochem Mol Biol, 2005, 21(1):53-60.

[2] 李华青,朱乃硕.重组乙肝表面抗原结合蛋白(SBP) 的制备及功能研究.复旦大学学报(自然科学版),2007,46(3): 390-393. Li H Q, Zhu N S. Preparation of the HBV surface antigen binding protein and the functional study. J Fudan Univ Natur Sci, 2007, 46(3): 390-393.

[3] 庞云,龚立,朱乃硕.一种乙肝表面抗原结合蛋白可作为疫苗佐剂.中国生物化学与分子生物学报,2009,25(11): 1024-1029. Pang Y, Gong Li,Zhu N S.An HBsAg binding protein expressed in Pichia pastoris can be used as an HBV vaccine adjuvant.Chinese Journal of Biochemistry and Molecular Biology,2009,25(11): 1024-1029.

[4] 庞云,龚立,彭思扬,等.乙肝表面抗原结合蛋白SBP在毕赤酵母中的表达纯化及功能鉴定. 生物工程学报,2009,25(10): 1564-1571. Pang Y, Gong L,Peng S Y, et al. Expression, purification and characterization of HbsAg binding protein in Pichia pastoris.Chin J Biotech,2009, 25(10): 1564-1571.

[5] 李津,范长胜,朱乃硕.表达乙肝表面抗原结合蛋白的毕赤酵母工程菌遗传稳定性研究.药物生物技术,2014,21(1):18-21. Li J,Fan C S, Zhu N S. Study on the genetic stability of the recombinant Pichia pastoris strain expressing HBsAg binding protein. Pharmaceutical Biotechnology,2014,21(1): 18-21.

[6] Cregg J M, Vedvick T S, Raschke W C. Recent advances in the expression of foreign genes in Pichia pastoris.Biotechnology(N Y),1993,11(8): 905-910.

[7] Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris.FEMS Microbiol Rev,2000,24(1): 45-66.

[8] Ottone S, Nguyen X, Bazin J, et al. Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis.Protein Expr Purif, 2007, 56(2): 177-188.

[9] 林俊涵.毕赤酵母高密度发酵工艺的研究.中国生物工程杂志,2009,29(5):120-125. Lin J H.High density fermentation control of Pichia pastoris.China Biotechnology,2009,29(5):120-125.

[10] 杨坤宇,何芳萍,李少伟,等.重组毕赤酵母高密度发酵表达H5N1禽流感病毒糖蛋白.生物工程学报,2009,25(5):773-778. Yang K Y, He F P, Li S W,et al. Expression of H5N1 avian influenza virus haemagglutinin protein in Pichia pastoris by high-density cell fermentation.Chin J Biotech,2009,25(5): 773-778.

[11] 王芸,华兆哲,刘立明,等.重组毕赤酵母高密度发酵生产碱性果胶酶的策略.生物工程学报,2003,24(4):635-639. Wang Y, Hua Z Z, Liu L M, et al. High-level production of alkaline polygalacturonate lyase in recombinant Pichia pastoris.Chin J Biotech,2003,24(4):635-639.

[12] 李洪淼,王红宁,许钦坤.毕赤酵母高密度发酵研究进展.生物技术通讯,2005,16(2):210-212. Li H M, Wang H N, Xu Q K. Advance of Pichia pastoris on high-density fermentation.Lett Biotechnol, 2005,16(2):210-212.

[13] 周祥山, 范位民, 张元兴. 不同甲醇流加策略对重组毕赤酵母高密度发酵生产水蛭素的影响.生物工程学报,2002, 18(3): 348-351. Zhou X S, Fan W M, Zhang Y X. Effects of different methanol feeding strategy on hirudin production in high-density fermentation by recombinant Pichia pastoris.Chin J Biotech, 2002, 18(3): 348-351.

[1]	高寅岭,张凤娇,赵贵众,张宏森,王风芹,宋安东. 衣康酸发酵研究进展[J]. 中国生物工程杂志, 2021, 41(5): 105-113.
[2]	陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.
[3]	陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[4]	石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 72-79.
[5]	杨娜,吴群,徐岩. 解淀粉芽孢杆菌合成surfactin的发酵策略优化 ^*[J]. 中国生物工程杂志, 2020, 40(7): 51-58.
[6]	王泽建,栗波,王萍,张琴,杭海峰,梁剑光,庄英萍. 葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响^*[J]. 中国生物工程杂志, 2020, 40(5): 30-39.
[7]	刘珍珍,田大勇. 狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 ^*[J]. 中国生物工程杂志, 2020, 40(4): 25-33.
[8]	王蒙,张全,高慧鹏,关浩,曹长海. 生物发酵法制备木糖醇的研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(3): 144-153.
[9]	王宝石,谭凤玲,李林波,李志刚,孟丽,邱立友,张明霞. 生物处理策略改善麸皮酚类化合物的生物可及性^*[J]. 中国生物工程杂志, 2020, 40(12): 88-94.
[10]	田园,李艳玲. 基于重组毕赤酵母的fusaruside生物合成 ^*[J]. 中国生物工程杂志, 2019, 39(7): 8-14.
[11]	彭强强,刘启,徐名强,张元兴,蔡孟浩. 新型重组毕赤酵母产人胰岛素前体的表达工艺研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 48-55.
[12]	王鑫淼,张康,陈晟,吴敬. 嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化 ^*[J]. 中国生物工程杂志, 2019, 39(7): 24-31.
[13]	严建,贾禄强,丁健,史仲平. 甲醇周期诱导控制强化毕赤酵母生产猪α干扰素 ^*[J]. 中国生物工程杂志, 2019, 39(6): 32-40.
[14]	姚银,闵琪,熊海容,张莉. 木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析 ^*[J]. 中国生物工程杂志, 2019, 39(3): 37-45.
[15]	张文玉,魏东升,钱江潮. 共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 24-33.

Viewed

Full text

Abstract

Cited

Shared

Discussed