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Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae

  • Long Ma
  • Xian Zhang
  • Zhihong Hu
  • Bin He
  • Mingqiang Ai
  • Bin ZengEmail author
Biotechnology and Industrial Microbiology - Research Paper
  • 11 Downloads

Abstract

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Keywords

Aspergillus oryzae Oxysterol-binding protein Ergosterol Microscale thermophoresis binding assay 

Notes

Funding information

This work was supported by National Natural Science Foundation of China (NSFC) (Grant Nos. 31460447 and 31700068), Natural Science Foundation of Jiangxi Province (20171BAB214004 and 20181BAB214001), and Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant Nos. GJJ170660, GJJ180600, and GJJGJJ170692).

Supplementary material

42770_2019_60_MOESM1_ESM.docx (29 kb)
Table S1 Sequence information used for phylogenetic analysis. (DOCX 29 kb)
42770_2019_60_MOESM2_ESM.docx (16 kb)
Table S2 Primers used in the qRT-PCR experiment. (DOCX 15.9 kb)
42770_2019_60_MOESM3_ESM.docx (12 kb)
Table S3 Sequence information for OSBP homologs used in multiple alignments of the ORDs. (DOCX 12 kb)

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Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Long Ma
    • 1
  • Xian Zhang
    • 1
  • Zhihong Hu
    • 1
  • Bin He
    • 1
  • Mingqiang Ai
    • 1
  • Bin Zeng
    • 1
    Email author
  1. 1.Jiangxi Key Laboratory of Bioprocess Engineering, College of Life SciencesJiangxi Science & Technology Normal UniversityNanchangChina

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