Expression,Purification,and Crystallization of Pif1 Helicase from Bacteroides fragilis

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

Abstract

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

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.

Figures/Tables 9

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

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）

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）

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

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

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

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

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

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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