Purification and Crystallization of PcCRN20-C from <i>Phytophthora capsici</i>

doi:10.13523/j.cb.1906036

China Biotechnology

2020, Vol. 40

Issue (1-2): 116-123 DOI: 10.13523/j.cb.1906036

Orginal Article

Purification and Crystallization of PcCRN20-C from Phytophthora capsici

ZHU Tong-tong¹,YANG Lei¹,LIU Ying-bao¹,SUN Wen-xiu^1,^**(

),ZHANG Xiu-guo²

1 College of Life Science,Yangtze University,Jingzhou 434000,China
2 College of Plant Protection,Shandong Agricultural University,Tai’an 271018,China

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Abstract

Crinkling and necrosis-inducing protein is a class of special secreted cytoplasmic protein when phytophthora occurs, which interferes with the normal physiological metabolism and function of host cells. However, the relationship between its three-dimensional structure and infection mechanisms need to be further studied. The sequence of PcCRN20-C was amplified by PCR from cDNA of Phytophthora capsici LT1534, which is 783bp in length and encodes 261 amino acids. The expression vector of pET-28a-MBP-PcCRN20-C was constructed and transformed to Escherichia coli BL21(DE3). The recombinant protein was induced under optimal condition, subsequently purified by nickel chelate affinity chromatography, ion-exchange chromatography, molecular-exclusion chromatography and trypsin hydrolysis technology. The product identified as a specific band of 25kDa by SDS-PAGE, which is the same as predicted. Subsequently, crystals were grown with the sitting-drop vapour-diffusion method and diffracted by X-ray. As a result, the crystals of CRN were obtained in a way of protein crystallography, which set a foundation for further study on the connect of CRN structure with function to analyze the pathogenic mechanism of P. capsici at the molecular level.

Key words： Phytophthora capsici Crinkling and necrosis-inducing protein Purification crystallization X-ray diffraction

Received: 26 June 2019 Published: 27 March 2020

ZTFLH:

S432.1

Corresponding Authors: Wen-xiu SUN E-mail: wenxiusun@163.com

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	Tong-tong ZHU
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	Xiu-guo ZHANG

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ZHU Tong-tong,YANG Lei,LIU Ying-bao,SUN Wen-xiu,ZHANG Xiu-guo. Purification and Crystallization of PcCRN20-C from Phytophthora capsici. China Biotechnology, 2020, 40(1-2): 116-123.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1906036 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I1-2/116

Fig.1 PCR amplification of PcCRN20-C gene fragment M:DNA ladder; 1-6:Amplification products of PcCRN20-C gene fragment by PCR

Fig.2 The nucleotide and deduced amino acid sequence of PcCRN20-C gene fragment The box indicates typical “DXS” domain

Fig.3 SDS-PAGE of PcCRN20-C after nickel chelate affinity chromatography M:Marker;1:Supernatant;2:Precipitant;3:Flowthrough;4:Washing buffer;5:Eluthion buffer;6:Resin after elution

Fig.4 Purification of PcCRN20-C (a)Purification of PcCRN20-C-MBP by anion exchange chromatography (b)Purification of PcCRN20-C -MBP by molecular-exclusion chromatography (c)Purification of PcCRN20-C following trypsin proteolysis by molecular-chromatography

Fig.5 SDS-PAGE analysis of purified PcCRN20-C (a)Purified PcCRN20-C-MBP by molecular-exclusion chromatography M: Marker; 1: Purified PcCRN20-C with MBP tag (b)Purified PcCRN20-C following trypsin proteolysis by molecular-chromatography M: Marker;1:Total protein after trypsin hydrolysis; 2-5: PcCRN20-C;6-10: MBP tag

Table 1 The result of PcCRN20-C mass spectrometry

Fig.6 PcCRN20-C crystals under different conditions

Fig.7 Crystal and diffraction pattern of PcCRN20-C (a)Crystal of PcCRN20-C (b) Diffraction pattern of PcCRN20-C


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