融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

doi:10.7503/cjcu20131071

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (2): 303.doi: 10.7503/cjcu20131071

融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

侯玥¹, 孙非¹, 金倞¹, 刘志屹¹, 郑清川², 梁重阳¹()

1. 吉林大学药学院, 长春 130021
2. 理论化学计算国家重点实验室, 长春 130021

收稿日期:2013-11-04 出版日期:2014-02-10 发布日期:2013-12-30
作者简介:联系人简介: 梁重阳, 男, 博士, 副教授, 主要从事基因工程药物方面的研究. E-mail:liang@jlu.edu.cn
基金资助:
国家自然科学基金(批准号: 81202446)和吉林省科技发展计划项目(批准号: 20090941)

Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris^†

HOU Yue¹, SUN Fei¹, JIN Jing¹, LIU Zhiyi¹, ZHENG Qingchuan², LIANG Chongyang^1,^*()

1. School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
2.State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130021, China

Received:2013-11-04 Online:2014-02-10 Published:2013-12-30
Contact: LIANG Chongyang E-mail:liang@jlu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.81202446) and the Technology Project of Jilin Province, China(No.20090941)

摘要/Abstract

摘要：

将灵芝免疫调节蛋白(LZ-8)与免疫球蛋白G的Fc片段(IgG Fc)进行融合表达, 通过Protein A亲和层析结合Superdex 75分子筛层析获得了高纯度LZ8-Fc. 凝血活性实验结果表明, LZ-8与Fc融合表达在一定程度上影响了其免疫活性; 利用分子动力学和圆二色光谱等技术研究了LZ8-Fc的蛋白折叠及组装方式, 发现LZ8-Fc的组装方式与LZ-8相同, 但是N末端的LZ-8可能影响下游IgG Fc片段的正确折叠, 表明在融合蛋白制备过程中, 位于N末端的蛋白对下游蛋白的正确折叠起到重要作用, 这为融合蛋白制备技术的优化及应用过程中的稳定提供了重要的理论依据.

关键词: LZ8-Fc, 分子动力学, 二聚体

Abstract:

Ganoderma lucidum immunoregulatory protein(LZ-8) was recombinant expressed in Pichia pastoris by prolonging its in vivo half-life through genetic fusion with human IgG-Fc fragment. High purify of LZ8-Fc was obtained by affinity chromatography and size-exclusion chromatography. The result of hemagglutination reaction indicated that LZ-8’s immunoregulatory activity was inhibited by IgG Fc fragment. Molecular dyna-mics simulation combined with SEC-HPLC and CD proved that LZ8-Fc assembled itself in the same way with LZ-8, dimer in natural state. All of the results suggested that LZ-8 in N-terminal affected IgG-Fc fragment in downstream, which is supposed an important issue in the recombinant expressing of fusion protein. The results will provide a solid basis for preparing and optimizing technology of fusion protein.

Key words: LZ8-Fc, Molecular dynamics, Dimer

中图分类号:

O629
O641

TrendMD:

侯玥, 孙非, 金倞, 刘志屹, 郑清川, 梁重阳. 融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响. 高等学校化学学报, 2014, 35(2): 303.

HOU Yue, SUN Fei, JIN Jing, LIU Zhiyi, ZHENG Qingchuan, LIANG Chongyang. Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris^†. Chem. J. Chinese Universities, 2014, 35(2): 303.

图/表 7

Fig.1 Construction and identification of pPICZαA-LZ8-Fc(A) Schematic of the pPICZαA-LZ8-Fc. LZ8-Fc sequence was cloned into pPICZαA via two restriction enzyme sites, EcoR I and Xho I, which next to α-factor secretion signal.(B) Identification of the pPICZαA-LZ8-Fc by PCR. Lane M: DNA marker; lanes 1 and 2: samples from the same PCR conditions with different tubes. The arrow points out the location of LZ8-Fc sequence.

Fig.2 SDS-PAGE(A), Western blotting(B) and ESI-TOF/TOF analysis(C) of LZ8-Fc expressed in P. pastoris strains X33(A) Lane M: protein molecular weight marker; lane 1: serum albumin; lane 2: LZ-8 protein; lanes 3—9: samples taken from the culture supernatant induced for 48, 42, 36, 30, 24, 12 and 6 h, respectively, with 0.5% methanol and separated by 0.12 mg/mL SDS-PAGE gel. (B) Lanes 1—7 were the same sequences of those in lanes 3—9 in (A). (C) Red fold words mean high abundance.

Fig.3 Two-steps chromatography process(A,B), SDS-PAGE(C) and IEF analysis(D) for LZ8-Fc(A, B) The arrows point to the peak of LZ8-Fc; (C) the single band(arrow) shows the same position with LZ8-Fc; (D) the band of LZ8-Fc(the arrow) shows the same position with the marker(pI=7.0).

Fig.4 Hemagglutination assay of LZ8-Fc and LZ-8LZ8-Fc and LZ-8 were diluted from 50 μg/mL to 1 μg/mL(row 1 to 8), columns a and b: LZ8-Fc; columns c and d: LZ-8.

Fig.5 SEC-HPLC analysis of LZ8-Fc’s assembling model(A) Purity analysis of the LZ8-Fc by HPLC. (B) Peaks 1—5 showed the absorption of the standard proteins, the molecular weights are 670, 158, 44, 17 and 1.35, respectively.

Fig.6 Western blotting(A), structure model(B) and molecular dynamics analysis result of bond length(2000 ps) for LZ8-Fc(A) Lane 1: LZ8-Fc concentration of 0.3 mg/mL; lane 2: concentrated LZ8-Fc was 2.5 mg/mL.(B) Structure model of LZ8-Fc, which connected with GGSS. (C, D) Distance of two peptides, which in (B) marked C and D.

Fig.7 CD spectrum(A) and analysis of secondary structure(B) of LZ8-Fc protein

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融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris^†

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融合蛋白表达过程中N末端蛋白对下游蛋白正确折叠的影响

Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris†

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Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris^†