豹蛙抗瘤酶与人血清白蛋白融合蛋白的毕赤酵母高效表达与活性测定

doi:10.13560/j.cnki.biotech.bull.1985.2015.10.034

生物技术通报 ›› 2015, Vol. 31 ›› Issue (10): 222-229.doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.034

豹蛙抗瘤酶与人血清白蛋白融合蛋白的毕赤酵母高效表达与活性测定

杨刚刚^1,3,马诚凯^1,3,史世会²,张全义²,王泽²,吕中原²,王绪洋²,许晓亚^1,3,崔晴晴^1,3,张继红^1,3,丁一^1,3,徐存拴^1,3

1. 河南师范大学生命科学学院, 新乡 453007;
2. 河南新乡华星药厂, 新乡 453007;
3.河南省一科技部共建细胞分化国家重点实验室培育基地和河南省生物工程重点实验室, 新乡 453007

收稿日期:2015-02-27 出版日期:2015-10-28 发布日期:2015-10-28
作者简介:杨刚刚, 男, 博士, 研究方向：微生物药物研究与开发; E-mail：yanggang1987@gmail.com
基金资助:
国家“973”前期研究专项（2012CB722304）, 河南省自然科学基金项目（142300413212, 132300413208）, 河南省重大科技攻关项目（111100910600）

Efficient Expression and Biological Activity Detection of Fusion Protein of Onconase with Human Serum Albumin in Pichia pastoris

Yang Ganggang^{1, 3}, Ma Chengkai^{1, 3}, Shi Shihui², Zhang Quanyi² , Wang Ze², Lü Zhongyuan², Wang Xuyang², Xu Xiaoya^{1, 3}, Cui Qingqing^{1, 3}, Zhang Jihong^{1, 3}, Ding Yi^{1, 3}, Xu Cunshuan^{1, 3}

1. College of Life Science, Henan Normal University, Xinxiang 453007; 2. Henan Xinxiang Hua Xing Pharmaceutical Factory, Xinxiang 453007; 3. State Key Laboratory Cultivation Base for Cell Differentiation Regulation and Henan Bioengineering Key Laboratory, Henan Normal University, Xinxiang 453007

Received:2015-02-27 Published:2015-10-28 Online:2015-10-28

摘要/Abstract

摘要： 豹蛙抗瘤酶（Onconase, ONC）, 又称豹蛙酶, 对多种肿瘤细胞和实体瘤具有显著杀伤作用, 为得到高表达的ONC, 利用HSA在毕赤酵母系统中的表达优势, 将HSA和ONC融合进行毕赤酵母表达并分离纯化。设计融合基因HSA-（Gly₄Ser₁）₃-ONC, 简称HSA-ONC, 构建至3种表达载体pPIC9、pPIC9K、pPICZα-A 并分别转染X-33、GS115和SMD1168 3种宿主菌, 在摇瓶和10 L规模优化表达条件和双水相偶联柱层析分离纯化目的蛋白并测定其生物学活性。结果显示, 在1 L摇瓶规模下, pPICZα-A/X-33/HSA-ONC组合的表达量优于其他组合, 且在pH7, 温度23℃条件下诱导10 d, 表达量达到最高（235 mg/L）。在10 L规模进行不同培养基条件的发酵, rHSA-ONC于pH 7的低盐基础盐培养基（Low salt basic salt medium, LS-BSM）, 甲醇浓度0.25%条件下诱导10 d, 表达量达到最高（2.02 g/L）。经双水相萃取偶联DEAE离子交换层析可得到纯度≥95%、收率高于70%的rHSA-ONC。生物活性检测发现, rHSA-ONC在体外能抑制多种癌细胞增殖。rHSA-ONC的表达量较rONC提高1倍（同等摩尔量情况下）, 同时在体外抑制多种癌细胞增殖。

关键词: 豹蛙抗瘤酶, 人血清蛋白, 毕赤酵母高效表达, 双水相萃取, 癌细胞活性

Abstract: Onconase（ONC, also known as ranpirnase or P-30 protein）has weak RNase activity and significant toxic effect on various tumor cells and solid tumors. In order to obtain ONC owing highly efficient expression, we utilized the advantage of human serum albumin（HSA）expression in Pichia pastoris, had a fusion of HSA and ONC which was expressed in P. pastoris, then separated and purified the expressed protein. We designed the fusion gene according to the amino acid sequence of HSA-ONC and synthesized it based on the codons of P. pastoris, HSA-ONC gene was inserted into vector of pPIC9, pPIC9K and pPICZα-A to form the recombinant plasmid pPIC9/HSA-ONC, pPIC9K/HSA-ONC and pPICZα-A/HSA-ONC, then the linearized recombinant plasmids were transformed into P. pastoris X-33, GSS115 and SMD1168 respectively. The expression conditions were optimized in the shake flask and 10-L bioreactor. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, and the biological activity was detected by SRB assay. The highest rHSA-ONC production reached 235 mg/L in pPICZα-A/X-33/HSA-ONC combination under the condition of pH7 and 23℃in 1-L flask after induced 10 d, better than other combinations. In the 10-L bioreactor, when the induction was performed in the low basic salt medium of pH 7, and induced 10 d under 0.25% ethanol, the highest rHSA-ONC production could reach 2.02 g/L. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, the purity was ≥ 95% and the yield was > 70%. The results of detecting biological activity showed that the rHSA-ONC inhibited the proliferation of various tumor cells in vitro. Conclusively, the expression level of rHSA-ONC was twice as that of rONC at the same molar dose, and rHSA-ONC inhibited the proliferation of various tumor cells in vitro.

Key words: onconase, human serum albumin, Pichia pastoris efficient expression, aqueous two-phase extraction, activity of tumor cells

杨刚刚,马诚凯,史世会,张全义,王泽,吕中原,王绪洋,许晓亚,崔晴晴,张继红,丁一,徐存拴. 豹蛙抗瘤酶与人血清白蛋白融合蛋白的毕赤酵母高效表达与活性测定[J]. 生物技术通报, 2015, 31(10): 222-229.

Yang Ganggang, Ma Chengkai, Shi Shihui, Zhang Quanyi , Wang Ze, Lü Zhongyuan, Wang Xuyang, Xu Xiaoya, Cui Qingqing, Zhang Jihong, Ding Yi, Xu Cunshuan. Efficient Expression and Biological Activity Detection of Fusion Protein of Onconase with Human Serum Albumin in Pichia pastoris[J]. Biotechnology Bulletin, 2015, 31(10): 222-229.

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豹蛙抗瘤酶与人血清白蛋白融合蛋白的毕赤酵母高效表达与活性测定

Efficient Expression and Biological Activity Detection of Fusion Protein of Onconase with Human Serum Albumin in Pichia pastoris

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