脆弱拟杆菌Pif1解旋酶的表达纯化与晶体生长

doi:10.13560/j.cnki.biotech.bull.1985.2020-1419

摘要/Abstract

摘要：

获得可用于X射线衍射的B.f Pif1单晶以用于探究B.f Pif1结构与功能。构建原核表达载体 pET15b-SUMO-B.f Pif1,并进行B.f Pif1重组蛋白的诱导表达;经镍柱亲和层析、SUMO酶切、DEAE交换层析与S200凝胶过滤层析等一系列纯化;并利用Stopped-flow技术检测纯化蛋白的活性;使用结晶机器人及多种试剂盒进行结晶条件筛选与优化培养,并进行初步的X射线衍射分析。获得高纯度（>98.5%）与高浓度（17 mg/mL）的B.f Pif1蛋白,动力学结果显示其解旋活性良好。结晶实验表明：在0.1 mol/L Bis-Tris乙酸（pH 8.3）,0.05 mol/L碳酸氢钠,5%甘油和0.015 mol/L亚精胺条件下培养出形态较好的单晶,其X射线衍射分辨率达到3.5 Å。成功表达纯化与结晶培养出具有较高分辨率的B.f Pif1蛋白的单晶。

关键词: Pif1解旋酶, 脆弱拟杆菌, 重组表达纯化, 蛋白结晶

Abstract:

To explore the structure and function of Pif1 helicase from Bacteroides fragilis,we obtained the monocrystal of B. f Pif1 for X-ray diffraction. The prokaryotic expression vector pET15B-SUMO-B.f Pif1 was successfully constructed,and the expression of recombinant B. fragilis Pif1 protein was induced. A series of protein purifications were performed,including Ni-NTA,SUMO-endonuclease digestion,DEAE ion-exchange column chromatography,and S200 gel filtration chromatography. The activity of the purified B. fragilis Pif1 protein was detected using the Stopped-Flow technique. Then,the crystallization by robot screening was performed with a variety of kits following by the optimization of crystal culture conditions,and the preliminary X-ray diffraction analysis was conducted. The B. fragilis Pif1 protein with high purity（>98.5%）and high concentration（17 mg/mL）was obtained. The kinetic results showed that purified protein had good Pif1-helicase activity. Crystallization experiments demonstrated that the monocrystals of B. fragilis Pif1 protein were obtained under the conditions of 0.1 mol/L Bis-Tris acetic acid（pH 8.3）,0.05 mol/L sodium bicarbonate,5% glycerol,and 0.015 mol/L spermidine,and its resolution of X -ray diffraction reached 3.5 Å. It is the first time to successfully express,purify and obtain the monocrystal of B. fragilis Pif1 protein with high X-ray diffraction.

Key words: Pif1 helicase, Bacteroides fragilis, recombinant expression and purification, protein crystallization

曹汝菲, 李泽轩, 许欢, 张莎, 张敏敏, 戴枫, 段晓雷. 脆弱拟杆菌Pif1解旋酶的表达纯化与晶体生长[J]. 生物技术通报, 2021, 37(9): 180-190.

CAO Ru-fei, LI Ze-xuan, XU Huan, ZHANG Sha, ZHANG Min-min, DAI Feng, DUAN Xiao-lei. Expression,Purification,and Crystallization of Pif1 Helicase from Bacteroides fragilis[J]. Biotechnology Bulletin, 2021, 37(9): 180-190.

图/表 9

图1 表达载体pET15b-SUMO-B.f Pif1的构建 A：pET15b-SUMO-B.f Pif1的示意图;B：通过PCR和限制性酶切鉴定重组载体。l：菌落PCR产物;2：对照质粒的酶切;3：NdeⅠ和EcoRⅠ对pET15b-SUMO-B.f Pif1的双酶切;M：DNA DS 5 000。红色箭头所指示靶标条带

Fig. 1 Construction of expression vector pET15b-SUMO-B.f Pif1 A：The schematic map of pET15b-SUMO-B.f Pif1. B：Identification of the recombinant vector by PCR and restriction enzyme digestion. Lane 1,the product of colony PCR;lane 2,the digestion of control plasmid;lane 3,the double digestion of pET15b-SUMO-B.f Pif1 by Nde I and EcoR I;M：DNA DS5 000. Target bands indicated by red arrows

图2 不同IPTG浓度与不同诱导温度诱导B.f Pif1蛋白的表达 A：不同IPTG浓度下诱导B.f Pif1表达的蛋白电泳图,1-4分别为经0.1、0.3、0.5和0.8 mmol/L IPTG诱导后的蛋白上清（4泳道对应诱导蛋白由于上样缓冲液更换而变模糊）;B：加IPTG后在不同诱导温度下诱导B.f Pif1表达的蛋白电泳图,1-3分别对应37℃诱导表达4 h、28℃诱导表达8 h、18℃诱导表达16 h）诱导后的蛋白上清（红色箭头指示目的蛋白）

Fig. 2 Expression of B.f Pif1 protein induced with different IPTG concentrations and different induction temperatures A：SDS-PAGE of B.f Pif1 expression induced with different IPTG concentrations. 1-4 lanes were protein supernatants after induction with 0.1,0.3,0.5,and 0.8 mmol/L IPTG（lane 4 corresponds to the induced protein obscured by loading buffer change）. B：SDS-PAGE of B.f pif1 expression induced with different induction temperatures, 1-3 lanes correspond to protein supernatants were inducted at 37℃ for 4 h,28℃ for 8 h,and 18℃ for 16 h,respectively（red arrows indicate target proteins of interest）

图3 B.f Pif1蛋白的诱导表达、镍柱纯化与SUMO酶切 A：B.f Pif1诱导表达破菌后沉淀、上清与穿出液的蛋白电泳图;B：第一次过Ni-NTA漂洗与不同浓度咪唑洗脱液的蛋白电泳图（1-2为Wash接收液,3-6为Elute接收液,其中4、5对应300 mmol/L咪唑洗脱管）;C：SUMO酶切蛋白标签前后的电泳对比图;D：第二次过Ni-NTA后上清液及沉淀电泳图（红色箭头指示目的蛋白,绿色箭头指示杂蛋白）

Fig. 3 Induced expression,Ni-NTA purification and SUMO digestion of B.f Pif1 protein A：SDS-PAGE analysis of the pellets,supernatants,and out flows of B.f Pif1 induced expression after bacterial ultrasonic crushing;B：SDS-PAGE of the first Ni-NTA purification with different concentrations of imidazole eluate buffer（1-2 lanes were wash tubes and 3-6 lanes were elute tubes,in which 4 and 5 lanes correspond to 300 mmol / L imidazole elution tubes;C：SDS-PAGE before and after SUMO digestion of the protein tags in B.f Pif1;D：SDS-PAGE of the second Ni-NTA purification with the supernatant and precipitate（red arrows indicated the target protein and green arrows indicated the hybrid protein）

图4 B.f Pif1蛋白的DEAE离子交换层析与S200分子筛纯化 A：重组B.f Pif1蛋白经DEAE离子交换层析的蛋白吸收峰图与SDS-PAGE电泳图,A峰与B峰为2个不同的电导率下洗脱峰;B：重组B.f Pif1蛋白经S200分子筛层析的蛋白吸收峰图与SDS-PAGE电泳图;C：最终蛋白纯化产物浓缩至17 mg/mL点样1 μL的SDS-PAGE电泳图

Fig. 4 Purifications of B.f Pif1 protein by DEAE ion-exchange chromatography and S200 gel filtration chromatography A：The absorption peak pattern and SDS-PAGE of the recombinant B.f Pif1 protein by DEAE ion-exchange chromatography. Peak A and B were eluted peaks at 2 different conductivities. B：The absorption peak pattern and SDS-PAGE of the recombinant B.f Pif1 protein by S200 gel filtration chromatography. C：SDS-PAGE of the final protein purified product with the concentration of 17 mg/mL by spotting 1μL of sample

图5 纯化后B.f Pif1蛋白具有良好的生物学活性 A：B.f Pif1蛋白解旋含G4 DNA与ss-dsdNA底物的活性对比;B：B.f Pif1解旋酶具有5'-3'的解旋极性

Fig. 5 Purified B.f Pif1 protein showing good biological activity A：Comparison of activity of B.f Pif1 protein unwinding G-quadruplex and ss-dsdNA substrate;B：B.f Pif1 helicase with good 5'-3' unwinding polarity

图6 结晶试剂盒初筛得到B.f Pif1晶体 A：Salt RxTMⅠ试剂盒35号条件筛选得到晶体;B：Cystal screenⅡ试剂盒74号条件筛选得到晶体;C：UY200紫外显微镜下观察Cystal screenⅡ试剂盒74号条件筛选出晶体的形态,其中绿色横线为晶体长宽测量标尺

Fig. 6 Protein crystals of B.f Pif1 preliminarily screened with different crystallization kits A：Crystals were obtained in the 35th condition of the Salt RxTMⅠ kit;B：crystals were obtained in the 74th condition of the Cystal screen II kit;C：ultraviolet microscope photograph of the selected crystals in the 74th condition of the Cystal screen II kit with UY200 microscope,of which the green lines were plotting scales

图7 B.f Pif1蛋白晶体培养条件的优化 A：采用悬滴法在显微镜下观察B.f Pif1孪晶逐渐变化;a-c：以B.f Pif1蛋白17 mg/mL为原浓度梯度稀释,d-f：逐步改变沉淀剂（不同PEG与亚精胺等）;B：采用座滴法在显微镜下观察B.f Pif 1单晶的逐渐生成

Fig. 7 Optimization of the culture conditions for B.f Pif1 protein crystals A：The gradual changes of B.f Pif1 twin-crystals were observed by microscope with hanging drop method;a-c subgraphs：gradient dilutions of B.f Pif1 protein 17 mg/mL（the original concentration）,d-f subgraphs：the change of precipitants with different PEG and spermidine;B：the gradual formation of B.f Pif 1 single crystal were observed by microscope with the sitting drop method

图8 B.f Pif1蛋白晶体生长情况晶体显微镜下观察到同一个液滴（坐滴法）中B.f Pif1蛋白晶体逐渐长大的照片;其左上角为从点样起观测的时间（单位为天数）：自左上到右下照片分别显示0、3、5、8、13和19 d时晶体的状态

Fig. 8 Growths of B.f Pif1 protein crystals A series of microscopic photographs of B.f Pif1 protein crystal growing status were observed in the same droplet with sitting drop method;the upper left corner was the initial observation time（unit：day）,and the photos from top left to bottom right showed the state of the crystals on 0,3,5,8,13 and 19 d respectively

图9 不同条件下B.f Pif1蛋白单晶的X射线衍射图谱及其对应蛋白电泳 A：在100 mmol/L NH4Acetate、16% PEG4000,pH 6.5条件下生长出来的单晶的X衍射及其蛋白电泳;B：在0.1 mol/L Bis-Tris乙酸（pH 8.3）、0.05 mol/L碳酸氢钠、5%甘油和0.015 mol/L亚精胺条件下生长出来的单晶的X衍射及其蛋白电泳

Fig. 9 X-ray diffraction patterns of B.f pif1 protein single crystal under different conditions and its corresponding SDS-PAGE A：X-ray diffraction and SDS-PAGE of the single protein crystal grown under the conditions of 100 mmol/L NH4Acetate,16% PEG4000 and pH 6.5;B：X-raydiffraction and SDS-PAGE of single crystals grown under the conditions of 0.1 mol/L mol/L Bis-Tris acetic acid（pH 8.3）,0.05 mol/L sodium bicarbonate,5% glycerol and 0.015 mol / L spermidine

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