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. 2017 Dec;7(6):527-531.
doi: 10.3892/br.2017.998. Epub 2017 Oct 10.

Molecular cloning, expression, purification and functional characterization of an antifungal cyclophilin protein from Panax ginseng

Hui Zhang  1 , Jiawen Wang  1 , Shuaijun Li  1 , Siming Wang  1 , Meichen Liu  1 , Weinan Wang  2 , Yu Zhao  1
Affiliations

Molecular cloning, expression, purification and functional characterization of an antifungal cyclophilin protein from Panax ginseng

Hui Zhang et al. Biomed Rep. 2017 Dec.

Abstract

Cyclophilins (CyPs), a member of peptidyl-prolyl cis-trans isomerases (PPIases), are ubiquitously distributed in organisms such as bacteria, yeast, plants and animals. CyPs have diverse biological functions, with some exhibiting antifungal and antiviral activities. In this study, Panax ginseng cyclophilin (pgCyP), a novel gene encoding an antifungal protein from Panax ginseng, was cloned, and its protein product was expressed in Escherichia coli, and then fractionated by affinity chromatography. The open reading frame of the pgCyP full-length coding sequence was found to encode a single-domain CyP-like protein of 174 amino residues with a calculated molecular weight of 18.7 kDa. The pGEX system was used to express pgCyP fused to glutathione S-transferase. After affinity purification, the protein showed a strong fungal resistance effect on Phytophthora cactorum. In addition, pgCyP showed high PPIase activity. To the best of our knowledge, the present study is the first successful effort to clone and characterize a CyP-like protein gene from Panax ginseng.

Keywords: Panax ginseng; antifungal activity; cyclophilin; expression and purification; peptidyl-prolyl cis-trans isomerase.

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Figures

Figure 1.
Figure 1.
Detection of the pgCyP gene transcript and sequence analysis. (A) PCR amplification of pgCyP by RT-PCR from total mRNA of ginseng leaves. Marker refers to the DNA molecular marker, while control is the mock-DNA negative control. (B) Schematic representation of constructs of GST-pgCyP-His6. (C) Predicted 3D structure model of pgCyP. The 3D structure model was predicted using the SWISS-MODEL server. (D) Alignment of the amino acid sequences of CyPs of Panax ginseng and other species. Residues comprising the divergent loop are shown in a black box, whereas the conserved Cys residues (Cys-40 and Cys-168) and Glu (Glu-83) are indicated with black arrows. pgCyP, Panax ginseng cyclophilin; PCR, polymerase chain reaction; GST, glutathione S-transferase; RT-PCR, reverse transcription-polymerase chain reaction; CyP, cyclophilin; ca, Chinese cabbage; cs, Citrus sinensis; zj, Ziziphus jujuba; rc, Ricinus communis.
Figure 2.
Figure 2.
Phylogenetic tree of cyclophilin proteins from plants, humans and yeast. The software program MEGA5 was used for alignment of amino acid sequences and to generate a phylogenetic tree based on the neighbor-joining method.
Figure 3.
Figure 3.
Expression, purification and refolding of GST-pgCyP-His6. Samples collected after purification of GST-pgCyP-His6 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining. Lane 1, marker; lane 2, sample before IPTG induction; lane 3, sample subjected to IPTG induction; lane 4, soluble-protein fraction; lane 5, insoluble-protein fraction; lane 6, solubilized denatured insoluble-protein fraction; lane 7, purification flow-through fraction; lane 8, purification wash fraction; lane 9, purification elution fraction with 100 mM imidazole. GST, glutathione S-transferase; pgCyP, Panax ginseng cyclophilin; IPTG, isopropyl β-D-1-thiogalactopyranoside.
Figure 4.
Figure 4.
Antifungal activity and peptidyl-prolyl cis-trans isomerase activity of pgCyP. (A) Antifungal activity of purified pgCyP and GST protein tested against Phytophthora cactorum at concentrations of 2.14 µM (GST), 1.28 µM (pgCyP) and 2.14 µM (pgCyP). (B) Growth inhibition of fungal pathogen by purified pgCyP. (C) Spectrophotometric assay performed with pgCyP or GST or in the absence of the two proteins. The graph is representative of at least three independent experiments. pgCyP, Panax ginseng cyclophilin; GST, glutathione S-transferase.
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