Expression, purification and characterization of ProL protein in the endophytic fungus <i>Cytospora rhizophorae</i> from <i>Morinda officinalis</i>

KONG Yali; YE Wei; LI Saini; ZHU Muzi; ZHONG Guohua; ZHANG Weimin

doi:10.7671/j.issn.1001-411X.201912016

Journal of South China Agricultural University > 2020 > 41(4): 82-89. > DOI: 10.7671/j.issn.1001-411X.201912016

KONG Yali, YE Wei, LI Saini, et al. Expression, purification and characterization of ProL protein in the endophytic fungus Cytospora rhizophorae from Morinda officinalis[J]. Journal of South China Agricultural University, 2020, 41(4): 82-89. DOI: 10.7671/j.issn.1001-411X.201912016

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Expression, purification and characterization of ProL protein in the endophytic fungus Cytospora rhizophorae from Morinda officinalis

KONG Yali^{1, 2,},
YE Wei²,
LI Saini²,
ZHU Muzi²,
ZHONG Guohua^1, ,,
ZHANG Weimin^2, ,

1.
College of Agriculture, South China Agricultural University/Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture and Rural Affairs/ Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, Guangzhou 510642, China
2.
Guangdong Institute of Microbiology, Guangdong Academy of Sciences/State Key Laboratory of Applied Microbiology Southern China/ Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application/ Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China

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Received Date: December 16, 2019
Available Online: May 17, 2023

Graphical Abstract

Abstract

Abstract

Objective
To clone the coding sequence of proL gene in the endophytic fungus Cytospora rhizophorae derived from Morinda officinalis, obtain ProL protein by heterologous expression and investigate its physicochemical properties, thereby providing a basis for the subsequent research on biological function of the ProL in the biosynthesis pathway of new bioactive compounds cytorhizins.

Method
The proL gene from C. rhizophorae was amplified by PCR, the proL gene fragment was inserted into the prokaryotic expression vector of pET28a by the homologous recombination method and heterologously expressed in Escherichia coli. The ProL protein was renatured by refolding buffer containing urea with a gradiently decreased concentration, and SDS-PAGE analysis and mass spectrometry sequencing were used to verify the target ProL protein. The bioinformatic methods were employed to analyze the similarity of ProL protein with other related proteins, and predict the structure and function of ProL protein.

Result
The coding sequence of proL gene was cloned, the open reading frame of proL gene is 909 bp in length, which encodes 303 amino acids, the molecular formula of ProL is C₁₄₉₅H₂₃₂₀N₄₂₄O₄₄₄S₁₃, the relative molecular weight is 33 754.22, the total number of atoms is 4 696, PI is 5.69, so ProL is an acidic protein. ProL protein was abundantly expressed as inclusion bodies in E.coli, the recombinant ProL was obtained with a purity of 98.9%. Bioinformatics analysis results indicated that ProL protein had the highest amino acid sequence similarity (59.40%) with amidohydrolase 2 from Aspergillus ibericus XP025570169.1. The three-dimensional structure model of ProL protein is composed of eight α-helixes and eight β-folds. The conserved amino acid sequence is located at the position from 207 to 216.

Conclusions
ProL protein belongs to the amidohydrolase superfamily, and is predicted as a novel protein. ProL protein might play a role of hydrolysis in the biosynthesis pathway of highly oxidized benzophenones.
- Morinda officinalis,
- Cytospora rhizophorae,
- proL gene,
- cloning,
- expression,
- bioinformatic

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References

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Expression, purification and characterization of ProL protein in the endophytic fungus Cytospora rhizophorae from Morinda officinalis

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