重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究

doi:10.13523/j.cb.20180508

中国生物工程杂志

2018, Vol. 38

Issue (5): 56-65 DOI: 10.13523/j.cb.20180508

研究报告

重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究

张潘潘¹,许延吉¹,王之可²,刘晓²,李素霞^1,^*(

)

1 华东理工大学生物反应器工程重点实验室上海 200237
2 上海雅心生物技术有限公司上海 201108

High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in Pichia pastoris

Pan-pan ZHANG¹,Yan-ji XU¹,Zhi-ke WANG²,Xiao LIU²,Su-xia LI^1,^*(

)

1 East China University of Science and Technology,State Key Laboratory of Bioreactor Engineering,Shanghai 200237,China
2 Shanghai Yaxin Biotehnology Co. Ltd,Shanghai 201108,China

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摘要：

目的:研究R122位点突变重组猪胰蛋白酶,与野生型酶相比较,该位点对重组猪胰蛋白酶(RPT)性质的影响。方法:以毕赤酵母GS115作为表达宿主,对RPT、突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)进行表达及纯化。并对其性质和稳定进行对比研究。结果:重组胰蛋白酶及其突变体在毕赤酵母中均获得了高效表达。相对于RPT,突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)在以N-苯甲酰-L-精氨酸乙酯 (BAEE)为底物时,具有更强底物结合力,三者的米氏常数分别为18.8μmol/L、9.0μmol/L和11.0μmol/L;两突变体耐高温耐碱能力增强;在Ca ²⁺存在及去除的条件下,突变体具有更强的抗自降解能力。结论:可以利用毕赤酵母高效表达重组胰蛋白酶及其突变体。mRPT(R122H)和mRPT(R122H/R73G/R130T) 相对于野生型RPT,对高pH条件和高温的耐受性增强,该稳定性的提高主要归因于R122位点的突变。

关键词： 重组猪胰蛋白酶; 毕赤酵母; 突变体; mRPT(R122H); mRPT(R122H/R73G/R130T); 性质; 稳定性

Abstract:

Objective:Study the effect of R122 residue mutation on the stability of recombinant porcine trypsin (RPT).Methods:RPT,mutants mRPT(R122H) and mRPT(R122H/R73G/R130) were expressed in Pichia pastoris GS115 and further purified. The properties and stabilities of RPT and two mutants was investigated and compared.Results:RPT and its mutants were highly expressed in Pichia pastoris. Relative to RPT, mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) showed higher affinity to substrate BAEE, The K_m values were 18.8μmol/L, 9.0μmol/L and 11.0μmol/L, respectively. Increased stability of mutants to high temperature and alkali were observed. And higher resistance against autolysis were got in the presence and without Ca ²⁺. Conclusion:Pichia pastoris can be used to efficiently express RPT and its mutants.Increased stability under alkaline condition and higher thermal stability and higher anti-self-digestion were got in mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) compared to wild-type RPT, which contribute to the site mutation at the R122.

Key words: Recombinant porcine trypsin Pichia pastoris Mutation mRPT(R122H) mRPT(R122H/R73G/R130T) Property Stability

收稿日期: 2018-01-11 出版日期: 2018-06-05

ZTFLH:

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通讯作者: 李素霞 E-mail: lisuxia@ecust.edu.cn

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引用本文:

张潘潘,许延吉,王之可,刘晓,李素霞. 重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究[J]. 中国生物工程杂志, 2018, 38(5): 56-65.

Pan-pan ZHANG,Yan-ji XU,Zhi-ke WANG,Xiao LIU,Su-xia LI. High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in Pichia pastoris. China Biotechnology, 2018, 38(5): 56-65.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180508 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I5/56

图1 猪胰蛋白酶三维结构模型模拟

图2 重组质粒的线性化

图3 重组猪胰蛋白酶原及其突变体诱导表达酶原

图4 mRPT(R122H) 10L发酵过程记录、生长曲线和SDS-PAGE表达鉴定

图5 mRPT(R122H)的纯化

图6 RPT和突变体mRPT(R122H)、mRPT(R122H/R73G/R130T)的Km值测定

表1 重组猪胰蛋白酶及两突变体的酶促动力学

图7 RPT和突变体mRPT(R122H)、 mRPT(R122H/R73G/R130T)的最适pH和pH稳定性

图8 重组猪胰蛋白酶和突变体mRPT(R122H)、mRPT(R122H/R73G/R130T)的最适温度

图9 RPT和突变体mRPT(R122H)、 mRPT(R122H/R73G/R130T)的温度稳定性

图10 RPT和突变体自降解活性测定

图11 RP-HPLC分析RPT及其突变体的自降解

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