Heterologous Expression and Purification of Recombinant CPS4 from

JIN Bao-long; CUI Guang-hong; DANG Bo-yue; LIU Shi-hui; QI Xiao-quan

doi:10.13422/j.cnki.syfjx.2014100094

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Heterologous Expression and Purification of Recombinant CPS4 from

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- Heterologous Expression and Purification of Recombinant CPS4 from
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 20, Issue 10, Pages: 94-98(2014)
- 作者机构：
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- DOI：10.13422/j.cnki.syfjx.2014100094
  CLC： R284.1;R285.5
- Published：2014
- 稿件说明：
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JIN Bao-long, CUI Guang-hong, DANG Bo-yue, et al. Heterologous Expression and Purification of Recombinant CPS4 from [J]. Chinese journal of experimental traditional medical formulae, 2014, 20(10): 94-98.
DOI：

JIN Bao-long, CUI Guang-hong, DANG Bo-yue, et al. Heterologous Expression and Purification of Recombinant CPS4 from [J]. Chinese journal of experimental traditional medical formulae, 2014, 20(10): 94-98. DOI： 10.13422/j.cnki.syfjx.2014100094.

摘要

目的：优化丹参柯巴基焦磷酸合酶基因家族新基因CPS4的原核表达体系并对重组蛋白进行纯化。方法：对2种原核表达菌株进行比较。对诱导条件，包括异丙基-β-D-硫代半乳糖苷（IPTG）诱导浓度、诱导时宿主菌的密度（A600）和诱导时间进行正交试验。利用HisTrap HP蛋白纯化柱对重组蛋白进行纯化。结果：Escherichia coli Tuner （DE3）表达菌株能诱导产生更多的可溶重组蛋白。最佳诱导表达条件为诱导时宿主菌的A600值0.7，IPTG浓度0.2 mmol·L-1，诱导表达时间10 h。HisTrap HP蛋白纯化柱纯化时梯度洗脱能得到纯度高的重组蛋白。结论：利用此优化体系可得到足量用于后续研究的重组蛋白，为其他二萜类合酶基因的原核表达提供参考。

Abstract

Objective: To find the optimal heterologous expression conditions and purification system for the new gene CPS4 from Salvia miltiorrhiza. Method: Compare two different prokaryotic expression strains and optimizing the induction conditions including A600 values of Escherichia coli

isopropyl-beta-D-galactose and glycosides(IPTG) concentration and induction time with orthogonal experiment. The recombinant protein was purified by HisTrap HP. Result: E. coli Tuner (DE3) induced more soluble recombinant protein than BL21 (DE3). The optimal expression conditions are A600 is 0.7

IPTG concentration is 0.2 mmol·L-1 and induction time for 10 h. The recombinant protein was purified by gradient elution with HisTrap HP. Conclusion: This optimal system can obtain enough protein for further study and provide a reference for the prokaryotic expression of the other diterpene synthase.

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