重组蛋白LuxS诱导表达及纯化条件的优化

doi:10.7506/spkx1002-6630-201519030

摘要/Abstract

摘要：

目的：优化重组蛋白LuxS诱导表达及纯化条件，提高具有生物活性的重组蛋白LuxS的表达量，为体外合成自诱导物2（autoinducer-2，AI-2）奠定基础。方法：采用单因素试验，优化诱导培养基、诱导温度、诱导前菌体生物量、诱导时间以及异丙基硫代半乳糖苷（isopropyl β-D-1-thiogalactopyranoside，IPTG）的浓度；采用Ni-NTAPurification System对重组蛋白进行纯化，比较Native Elution Buffer的咪唑浓度和pH值对重组蛋白纯化的影响，确定最佳的Native Elution Buffer；对比蛋白与树脂结合时不同缓冲液的纯化效果，选择最优的结合缓冲液。结果：重组蛋白的最佳诱导表达条件为：以LB培养基为诱导培养基，菌液OD600 nm为0.6～0.8，IPTG浓度为0.1 mmol/L，诱导温度为37 ℃，诱导时间为12 h。采用添加3 mol/L NaCl的磷酸盐缓冲液作为蛋白与树脂结合的缓冲液，含500 mmol/L咪唑的Native Elution Buffer（pH 6.5）作为蛋白洗脱液。使用分离获得的LuxS蛋白合成的AI-2生物活性约为阳性对照的6 倍。结论：本研究成功优化了重组蛋白LuxS的诱导表达及纯化条件，获得了具有生物活性的重组蛋白，并成功合成了AI-2。

关键词: 重组蛋白, LuxS, 诱导表达, 纯化, 自诱导物2

Abstract:

Objective: To optimize the expression and purification conditions of recombinant protein LuxS. Methods: The
induction conditions, including medium components, induction temperature, bacterial biomass before induction, induction
time and IPTG concentration were optimized by single-factor design. Recombinant protein was purified using Ni-NTA
purification system under native conditions. The optimum native elution buffer was determined by comparing the effects of
imidazole concentration and native elution buffer pH on purification efficiency. The optimum binding buffer for protein and
resin was determined by comparing the effects of different types of binding buffer on purification efficiency. Results: The
optimum conditions for expression of recombinant protein were determined as follows: when the OD600 nm reached 0.6–0.8,
using LB medium as the induction medium, the induction was initiated with 0.1 mmol/L IPTG at 37 ℃ for 12 h. PBS buffer
containing 3 mol/L NaCl was used for the binding of protein and resin. The recombinant protein was eluted with native elution
buffer (pH 6.5) containing 500 mmol/L imidazole. The biological activity of the synthesized AI-2 in vitro, which was produced
catalytically by Pfs and recombinant protein LuxS obtained in this study, was approximately 6-fold higher than that from the
positive control. Conclusion: The expression and purification conditions of recombinant protein LuxS were optimized, and the
recombinant protein LuxS with bioactivity was obtained. Finally, AI-2 was synthesized successfully in vitro.

Key words: recombinant protein, LuxS, induced expression, purification, autoinducer-2 (AI-2)

中图分类号:

TS201.3

杨杰，张筠，孟祥晨. 重组蛋白LuxS诱导表达及纯化条件的优化[J]. 食品科学, doi: 10.7506/spkx1002-6630-201519030.

YANG Jie, ZHANG Yun, MENG Xiangchen. Optimization of Expression and Purification Conditions of Recombinant Protein LuxS[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201519030.

[1]	项婷，夏琛，刘健华，王超然，沈建福. 蛹虫草中新化合物的分离纯化、结构鉴定及抗肿瘤活性[J]. 食品科学, 2021, 42(8): 235-242.
[2]	王伟，李津津，迟海. 解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理[J]. 食品科学, 2021, 42(7): 29-34.
[3]	魏晨业，包晓玮，王娟，何梦梦，曾兰君，张亚涛. 沙棘多糖分离纯化及抗氧化活性[J]. 食品科学, 2021, 42(4): 227-232.
[4]	胡晓，周雅，陈星星，李来好，杨贤庆，陈胜军，吴燕燕，杨少玲. 罗非鱼肌浆蛋白源抗氧化肽的制备、分离纯化及其体外抗氧化活性[J]. 食品科学, 2021, 42(3): 63-70.
[5]	高兆建，王秋芬，丁飞鸿，许祥，赵宜峰，焦魏，陈腾. 广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定[J]. 食品科学, 2021, 42(2): 143-150.
[6]	杨其乐，丁一峰，张宇，聂若男，李亚萌，王佳，王小红. 沙门氏菌噬菌体LPST144尾纤维gp38的序列分析及其结合活性[J]. 食品科学, 2021, 42(2): 66-73.
[7]	李钰景，孙丽平，庄永亮. 红毛丹果皮中老鹳草素的分离纯化及其热稳定性[J]. 食品科学, 2021, 42(15): 44-49.
[8]	高兆建，丁飞鸿，陈欢，耿云龙，赵宜峰. 产黄青霉抗真菌几丁质酶的纯化及特性分析[J]. 食品科学, 2021, 42(14): 129-136.
[9]	杨汝晴，陈守峰，肖琳琳，陈玉磊，孙乐常，张凌晶，刘光明，曹敏杰. 鲈鱼脯氨酰内肽酶的分离纯化、性质分析及分子克隆[J]. 食品科学, 2021, 42(14): 78-85.
[10]	沙玉欢，毛晓英，吴庆智，张建，程卫东. 核桃分心木黄酮物质的组分及其抗氧化性分析[J]. 食品科学, 2021, 42(12): 91-98.
[11]	崔晓颖，彭新颜，贺红军，张敏，张晓彤. 芋头多酚氧化酶的分离纯化与酶学特性[J]. 食品科学, 2021, 42(12): 107-115.
[12]	刘静波，王子秦，于一丁，张婷，刘博群. 豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析[J]. 食品科学, 2021, 42(12): 123-129.
[13]	高兆建，胡鑫强，宋玉林，丁飞鸿，赵宜峰，陈腾. 解淀粉芽孢杆菌I型耐热普鲁兰酶的纯化及酶特性分析[J]. 食品科学, 2021, 42(12): 130-137.
[14]	王思琪，胡彦波，翟丽媛，石曾卉，赵珺. 豆渣可溶酸性多糖的分离纯化及结构解析[J]. 食品科学, 2021, 42(10): 52-57.
[15]	陈宏，章骞，陈玉磊，翁凌，张凌晶，谢少浩，刘光明，曹敏杰. 利用牡蛎制备DPP-IV抑制肽及其活性分析[J]. 食品科学, 2021, 42(10): 120-126.

重组蛋白LuxS诱导表达及纯化条件的优化

Optimization of Expression and Purification Conditions of Recombinant Protein LuxS

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价