Purification and characterization of <i>Arabidopsis thaliana</i> oligosaccharyltransferase complexes from the native host: a protein super‐expression system for structural studies

Jeong, In Sil; Lee, Sang‐Min; Bonkhofer, Florian; Tolley, Jordan P.; Fukudome, Akihito; Nagashima, Yukihiro; May, Kimberly; Rips, Stephan; Lee, Sang Yup; Gallois, Patrick; Russell, William K.; Jung, Hyun Suk; Schaewen, Antje von; Koiwa, Hisashi

doi:10.1111/tpj.13847

Cited by 38 publications

(23 citation statements)

References 53 publications

(104 reference statements)

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“…Mammalian ribophorin I affected the glycosylation of different peptides . A previous analysis demonstrated that Arabidopsis has two OST1 subtypes which interact with STT3a . Our evolutionary analysis of OST1 revealed that plant OST1 has diverged into two conservatively evolved clades in vascular plants (Fig.…”

Section: Discussionmentioning

confidence: 64%

Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms

et al. 2020

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STT3 is a catalytic subunit of hetero‐oligomeric oligosaccharyltransferase (OST), which is important for asparagine‐linked glycosylation. In mammals and plants, OSTs with different STT3 isoforms exhibit distinct levels of enzymatic efficiency or different responses to stressors. Although two different STT3 isoforms have been identified in both plants and animals, it remains unclear whether these isoforms result from gene duplication in an ancestral eukaryote. Furthermore, the molecular mechanisms underlying the functional divergences between the two STT3 isoforms in plant have not been well elucidated. Here, we conducted phylogenetic analysis of the major evolutionary node species and suggested that gene duplications of STT3 may have occurred independently in animals and plants. Across land plants, the exon–intron structure differed between the two STT3 isoforms, but was highly conserved for each isoform. Most angiosperm STT3a genes had 23 exons with intron phase 0, while STT3b genes had 6 exons with intron phase 2. Characteristic motifs (motif 18 and 19) of STT3s were mapped to different structure domains in the plant STT3 proteins. These two motifs overlap with regions of high nonsynonymous‐to‐synonymous substitution rates, suggesting the regions may be related to functional difference between STT3a and STT3b. In addition, promoter elements and gene expression profiles were different between the two isoforms, indicating expression pattern divergence of the two genes. Collectively, the identified differences may result in the functional divergence of plant STT3s.

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Section: Discussionmentioning

confidence: 64%

Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms

et al. 2020

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“…Native and recombinant proteins are used in a wide range of areas in the biopharmaceutical, enzyme, and agricultural industries. Such molecules are increasingly being used in the fields of pharmacy, diagnostics, milk, diet, detergents, textiles, clothing, paper, pulp, polymers, and plastics [ 2 , 3 , 4 , 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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“…We recently reported an Arabidopsis-based recombinant protein production platform (super-expression system) suitable for biochemical and structural studies (Figure 1). 18 This platform yielded as much as 0.4 mg of purified protein per gram fresh weight (FW) using a heterologous model protein (mCherry) for expression and purification. The benefit as homologous overexpression system was demonstrated for oligosaccharyltransferase (OT), an integral-membrane protein complex consisting of seven or moresubunits that could be assembled on a single overexpressed central STT3a subunit.…”

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confidence: 99%

“…The OT-ribosome super-complex was then purified and its three-dimensional structure determined by transmission electronmicroscopy at 30Å resolution. 18 Combined with our pilot study applying this technology to a soluble enzyme (CTD phosphatase-like 4), 19 which was difficult to express in E. coli as an active form, we propose the Arabidopsis superexpression system as a versatile recombinant protein production platform.…”

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confidence: 99%

“…However, availability of many genotypes is another merit of Arabidopsis. Our current system uses rdr6-11 as the standard host to avoid gene silencing (similar to P19 co-expression in the Nb system), 18,22 but one can use various genotypes or mutant backgrounds to improve yield and activity of recombinant proteins, or study the role of a particular post-translational modification.…”

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confidence: 99%

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Improved recombinant protein production in Arabidopsis thaliana

Schaewen

Jeong

Rips

et al. 2018

Plant Signaling & Behavior

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Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super‐expression system for structural studies

Cited by 38 publications

References 53 publications

Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms

Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Improved recombinant protein production in Arabidopsis thaliana

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