Dataset Information

Protein characterization, purification, and sequence analysis data for plant-made catfish interleukin 22.

ABSTRACT: Production and purification of a novel protein in plants results in the generation of multiple data sets leading to an optimized protocol for recovering the recombinant protein. This article presents the data collected in the process used to produce, purify and validate a catfish interleukin 22 (cfIL-22) expressed using a plant-based platform. A commonly used workflow for confirming optimal expression and extraction of the recombinant protein was employed and is outlined herein. The complete research article, including activity analysis of plant-produced cfIL-22, is published in Journal of Biotechnology Elkins and Dolan [1]. Data collected in optimizing the expression, purification and characterization process of cfIL-22 includes stained protein gels and western immunoblot analyses, DNA and protein sequencing, post-translational modification predictions and protein structure predictions. The value of this data lies not only in future work in expressing interleukin 22 orthologs but also provides a guide for optimizing the production and validating similar complex animal/human proteins produced in plants.

SUBMITTER: Elkins L

PROVIDER: S-EPMC7804605 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Protein characterization, purification, and sequence analysis data for plant-made catfish interleukin 22.

Elkins Lana L Dolan Maureen C MC

Data in brief 20201221

Production and purification of a novel protein in plants results in the generation of multiple data sets leading to an optimized protocol for recovering the recombinant protein. This article presents the data collected in the process used to produce, purify and validate a catfish interleukin 22 (cfIL-22) expressed using a plant-based platform. A commonly used workflow for confirming optimal expression and extraction of the recombinant protein was employed and is outlined herein. The complete res ...[more]

PMID: 33490321

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Project description:The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14++/CD16+ intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response.

Dataset Information

Protein characterization, purification, and sequence analysis data for plant-made catfish interleukin 22.

Publications

Protein characterization, purification, and sequence analysis data for plant-made catfish interleukin 22.

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