Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

Bokhove, Marcel; Hosseini, Hamed Sadat Al; Saitô, Takako; Dioguardi, Elisa; Gegenschatz‐Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; Sanctis, Daniele de; Han, Ling; Jovine, Luca

doi:10.1016/j.jsb.2016.01.016

Cited by 42 publications

(45 citation statements)

References 45 publications

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“…The resulting PCR products, isolated after digestion with Not I and Xho I were subcloned in frame with the mMBP gene of pHLmMBP-115, a mammalian expression vector derived frompHLsec42. Restrictions enzymes were purchased from Fermentas (San Leon-Rot, Germany).…”

Section: Methodsmentioning

confidence: 99%

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

Algarra

Han

Soriano‐Úbeda

et al. 2016

Sci Rep

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OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg.

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Section: Methodsmentioning

confidence: 99%

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

Algarra

Han

Soriano‐Úbeda

et al. 2016

Sci Rep

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“…For analyzing the oligomerization state of egg coat protein precursors, medaka ZI-1,2 (which does not contain N-glycosylation sites) was expressed in HEK293T cells (DuBridge et al, 1987) , grown in DMEM medium supplemented with 4 mM L-Gln 2% fetal bovine serum and transiently transfected using 25 kDa branched PEI (Aricescu et al, 2006;Bokhove et al, 2016b) . GnTI-deficient HEK293S cells (Reeves et al, 2002) were used to express ZI-3 carrying Endoglycosidase H (Endo H)-sensitive Man5GlcNAc2 N-glycans.…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

“…GnTI-deficient HEK293S cells (Reeves et al, 2002) were used to express ZI-3 carrying Endoglycosidase H (Endo H)-sensitive Man5GlcNAc2 N-glycans. These carbohydrate chains were then enzymatically trimmed to single GlcNAc residues during protein purification, which was performed by batch immobilized metal ion affinity (IMAC) using nickel agarose slurry (Ni-NTA, QIAGEN or Ni Sepharose High Performance, GE Healthcare) and size-exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated against 20 mM HEPES pH 7.5, 150 mM NaCl (Bokhove et al, 2016b) .…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Stsiapanava

Brunati

et al. 2020

Preprint

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SUMMARYAssembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

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“…One approach to stabilize flexible proteins has been described by Moon and coworkers and utilizes maltosebinding protein (MBP) with surface-entropy reduction (SER) mutations as an N-terminal carrier protein to enhance protein crystallization (Moon et al, 2010). MBP has been reported to enhance the solubility of proteins when expressed as an N-terminal fusion in Escherichia coli (Waugh, 2016;Jin et al, 2017) and mammalian expression systems (Reuten et al, 2016;Bokhove et al, 2016), but to date this has not been reported for insect-cell expression systems. We describe the expression of the FXIIa protease domain as a secreted MBP fusion using DES, which facilitated the crystal structure determination of MBP-FXIIa His in the active conformation in complex with the peptidomimetic inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK).…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

Pathak

Manna

et al. 2019

Acta Cryst Sect D Struct Biol

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Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (FXIIa His ) with yields of $1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-FXIIa His ). Crystal structures were determined of MBP-FXIIa His in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of FXIIa His in isolation. The FXIIa His structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the FXIIa His structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with FXIIa are also described. These structural studies of FXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and antiinflammatory agents.

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Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

Cited by 42 publications

References 45 publications

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

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