An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system

Tabata, Sanae; Kitago, Yu; Fujii, Yuki; Mihara, Emiko; Tamura-Kawakami, Keiko; Norioka, Naoko; Takahashi, Kazuyuki; Kaneko, Mika K.; Kato, Yukinari; Takagi, Junichi

doi:10.1016/j.pep.2018.03.004

Cited by 9 publications

(5 citation statements)

References 19 publications

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“…The coding region of saposin A was cloned into a modified pET-28b vector that harbors an N-terminal His 6 tag followed by the 'eTEV' sequence (Tabata et al, 2018), resulting in the protein sequence MGSSHHHHHHSSGLVPRENLYFQGM GSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCD WLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCS ALNLCES. The protein cleaved by TEV protease represents the saposin A polypeptide sequence with three additional Nterminal residues (Gly-Met-Gly).…”

Section: Expression and Purification Of Saposin Amentioning

confidence: 99%

Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Nagamura

Fukuda

Kawamoto

et al. 2019

Acta Crystallogr F Struct Biol Commun

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Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1–H6 and H7–H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Å resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.

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Section: Expression and Purification Of Saposin Amentioning

confidence: 99%

Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Nagamura

Fukuda

Kawamoto

et al. 2019

Acta Crystallogr F Struct Biol Commun

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“…Preparation of PA-tagged mouse USAG-1 recombinant protein from mammalian cells was performed as previously reported (42). Other tagged USAG-1 recombinant proteins, derived from E. coli or baculoviral expression systems (R&D systems Inc., MN, USA; MyBiosource, CA, USA), were used for the production of antibodies, as antigens, and in the solid phase and/or sandwich enzyme-linked immunosorbent assay (ELISA).…”

Section: Plasmid and Recombinant Proteinsmentioning

confidence: 99%

“…Briefly, 5 g of purified anti-USAG-1 mAbs was incubated with 15 l of Protein A-Sepharose (GE Healthcare) for 2.5 hours at 15° to 25°C, followed by a brief wash with PBS. The beads were incubated with the culture supernatants of the Expi293F cells transiently transfected with either mouse or human USAG-1 containing N-terminal PA tag (42). After extensive washing with PBS, the bound proteins were eluted from the beads by adding SDS sample buffer and then analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) using 5 to 20% gradient gel under nonreducing conditions.…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…The soluble human LRP6 ectodomain fragments containing different regions (E1-E4, residues 1 to 1244; E1-E2, residues 1 to 629; E3-E4, residues 630 to 1244) were C-terminally His-tagged and transiently expressed in Expi293F cells as described previously (44). After immobilizing onto Ni-NTA beads, they were further incubated with the culture supernatants of the Expi293F cells stably expressing mouse USAG-1 established previously (42). The bound USAG-1 was eluted together with the LRP6 fragments by SDS and analyzed by nonreducing SDS-PAGE.…”

Section: Lrp6-binding Assaymentioning

confidence: 99%

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Anti–USAG-1 therapy for tooth regeneration through enhanced BMP signaling

et al. 2021

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Uterine sensitization–associated gene-1 (USAG-1) deficiency leads to enhanced bone morphogenetic protein (BMP) signaling, leading to supernumerary teeth formation. Furthermore, antibodies interfering with binding of USAG-1 to BMP, but not lipoprotein receptor–related protein 5/6 (LRP5/6), accelerate tooth development. Since USAG-1 inhibits Wnt and BMP signals, the essential factors for tooth development, via direct binding to BMP and Wnt coreceptor LRP5/6, we hypothesized that USAG-1 plays key regulatory roles in suppressing tooth development. However, the involvement of USAG-1 in various types of congenital tooth agenesis remains unknown. Here, we show that blocking USAG-1 function through USAG-1 knockout or anti–USAG-1 antibody administration relieves congenital tooth agenesis caused by various genetic abnormalities in mice. Our results demonstrate that USAG-1 controls the number of teeth by inhibiting development of potential tooth germs in wild-type or mutant mice missing teeth. Anti–USAG-1 antibody administration is, therefore, a promising approach for tooth regeneration therapy.

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“…The PA tag and NZ-1 antibody have a number of properties that are attractive as a purification handle, including a very high binding affinity relative to other purification tags (Table 2), mild elution conditions, and a very low dissociation rate that allows for extensive washing for removal of contaminants (Fujii et al 2014). As a purification tool, the PA tag/NZ-1 system has been used extensively to purify a wide range of proteins (Kitago et al 2015;Mihara et al 2016;Suzuki et al 2016;Umitsu et al 2016;Meng et al 2017;Nagae et al 2018;Tabata et al 2018).…”

Section: Pa Tag/nz-1 Antibody Systemmentioning

confidence: 99%

Advances in domain and subunit localization technology for electron microscopy

Brown

Takagi

2019

Biophys Rev

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The award of the 2017 Nobel Prize in chemistry, 'for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution', was recognition that this method, and electron microscopy more generally, represent powerful techniques in the scientific armamentarium for atomic level structural assessment. Technical advances in equipment, software, and sample preparation, have allowed for high-resolution structural determination of a range of complex biological machinery such that the position of individual atoms within these mega-structures can be determined. However, not all targets are amenable to attaining such high-resolution structures and some may only be resolved at so-called intermediate resolutions. In these cases, other tools are needed to correctly characterize the domain or subunit orientation and architecture. In this review, we will outline various methods that can provide additional information to help understand the macro-level organization of proteins/biomolecular complexes when highresolution structural description is not available. In particular, we will discuss the recent development and use of a novel protein purification approach, known as the the PA tag/NZ-1 antibody system, which provides numberous beneficial properties, when used in electron microscopy experimentation.

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An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system

Cited by 9 publications

References 19 publications

Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Anti–USAG-1 therapy for tooth regeneration through enhanced BMP signaling

Advances in domain and subunit localization technology for electron microscopy

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An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system

Cited by 9 publications

References 19 publications

Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2

Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2

Anti–USAG-1 therapy for tooth regeneration through enhanced BMP signaling

Advances in domain and subunit localization technology for electron microscopy

Contact Info

Product

Resources

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Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2