Dataset Information

Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans.

ABSTRACT: Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The neverland (NVD) family genes encode the conserved Rieske-type oxygenases, which are accountable for the dehydrogenation during the synthesis and regulation of steroid hormones. However, the His-tagged NVD protein from Caenorhabditis elegans expresses as inclusion bodies in Escherichia coli BL21 (DE3). This bottleneck can be solved through refolding by urea or the introduction of a maltose-binding protein (MBP) tag at the N-terminus. Through further research on purification after the introduction of a MBP tag at the N-terminus, the CD measurement and fluorescence-based thermal shift assay indicated that MBP was favorable for the NVD proteins' solubility and stability, which may be beneficial for the large-scale manufacture of NVD protein for further research. The structural model contained the Rieske [2Fe-2S] domain and non-heme iron-binding motif, which were similar to 3-ketosteroid 9 ?-hydroxylase.

SUBMITTER: Mao S

PROVIDER: S-EPMC7609953 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans.

Mao Shuhong S Song Zhan Z Wu Mian M Wang Xiaorui X Lu Fuping F Qin Hui-Min HM

Frontiers in bioengineering and biotechnology 20201021

Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The neverland (NVD) family genes encode the conserved Rieske-type oxygenases, which are accountable for the dehydrogenation during the synthesis and regulation of steroid hormones. However, the His-tagged NVD protein from Caenorhabditis elegans expresses as inclusion bodies in Escherichia coli BL21 (DE3). This bottleneck can be solved through refolding b ...[more]

PMID: 33195160

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Project description:BackgroundAffinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide.ResultsWe generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules.ConclusionsThe method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

Dataset Information

Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans.

Publications

Expression, Purification, Refolding, and Characterization of a <i>Neverland</i> Protein From <i>Caenorhabditis elegans</i>.

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