Bioinformatics analysis and enzymatic activity test of quiescin sulfhydryl oxidase from Plasmodium berghei

doi:10.12140/j.issn.1000-7423.2020.02.005

Abstract

Abstract:

Objective To clone and express the gene of Plasmodium berghei quiescin sulfhydryl oxidase (PbQSOX), purify the recombinant protein rPbQSOX, and analyze its bioinformatics characteristics and enzymatic activity.Methods The signal peptides, protein domains, active sites and homology of PbQSOX protein were analyzed by BLAST, SMART and ClustalW softwares. The phylogenetic tree of PbQSOX was constructed by BLAST and MAGA5. The tertiary structure of PbQSOX protein was predicted by SWISS-MODEL. Female BALB/c mice were intraperitoneally injected with 1 × 10 ⁷ P. berghei protozoan/mouse. On day 4 after infection, P. berghei genomic DNA was extracted. The PbQSOX gene was amplified by PCR. After confirming the product by sequencing, the target product was cloned into pET30a(+) vector to construct recombinant plasmid pET30a(+)-PbQSOX. The verified plasmid was transfected into E. coli BL-21 after induction by isopropyl β-D-thiogalactoside, the bacteria culture medium was analysed underwent by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The soluble recombinant PbQSOX (rPbQSOX) was purified by Ni-triacetate agarose column, and the enzyme activity was analyzed using TCEP [Tris(2-carboxyethyl)phosphine] or DL-Dithiothreitol (DTT) as the enzyme reaction substrate. Results BLAST analysis identified homologues of PbQSOX from other species. Multiple-sequence blasting and domain analysis showed that PbQSOX contained a signal peptide, and Trx1, ψErv and Erv/ALR domains, but lacked the Trx2 domain. The PbQSOX protein contained three key CXXC activation motifs (C, cysteine; X, any amino acid): CPAC, CRNC and CNYC. The phylogenetic tree constructed by MAGA5 using the neighbor joining method showed that the PbQSOX was genetically most close to the P. yoelii QSOX (PyQSOX). SWISS-MODEL analysis showed that the tertiary structure of PbQSOX can be predicted using known Trypanosoma brucei QSOX (TbQSOX). PCR amplification of PbQSOX resulted in a specific band of 1 486 bp, sequence of which was confirmed by sequencing. The PET30a (+) -PbQSOX plasmid was verified by sequencing after Nde Ⅰ and Hind Ⅲ digestion. SDA-PAGE and Western blotting analysis showed that rPbQSOX was expressed in soluble form with a relative molecular weight of about 59 000. The enzymatic activities of purified rPbQSOX toward TCEP and DTT were 0.84 ± 0.18 and 0.78 ± 0.14, respectively.Conclusion The expressed PbQSOX protein contains a single Trx1 domain. The rPbQSOX is a soluble protein that has some catalytic activity of the quiescin sulfhydryl oxidase.

Key words: Plasmodium, Bioinformatics, Protein expression, Plasmodium berghei quiescin sulfhydryl oxidase

CLC Number:

R382.319

Wen-qi ZHENG, Xue-min FENG, Ya-ming CAO, Yan-qiu HAN, Jun-rui WANG. Bioinformatics analysis and enzymatic activity test of quiescin sulfhydryl oxidase from Plasmodium berghei[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(2): 159-165.

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