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#23656633   2013/06/20 Save this To Up

Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization.

Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.

1329 related Products with: Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization.

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#22252869   2012/03/19 Save this To Up

Identification of the cellular receptor of Clostridium spiroforme toxin.

Clostridium spiroforme produces the binary actin-ADP-ribosylating toxin CST (C. spiroforme toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of C. spiroforme toxin in Bacillus megaterium. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins Clostridium difficile transferase (CDT) and Clostridium perfringens iota toxin. Microscopic studies revealed that CST, but not the related Clostridium botulinum C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with C. difficile CDT and C. perfringens iota toxin as a host cell surface receptor.

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#18397316   2008/04/30 Save this To Up

Interaction with model membranes and pore formation by human stefin B: studying the native and prefibrillar states.

Human stefin B, from the family of cystatins, is used as a model amyloidogenic protein in studies of the mechanism of amyloid fibril formation and related cytotoxicity. Interaction of the protein's prefibrillar oligomers/aggregates with predominantly acidic phospholipid membranes is known to correlate with cellular toxicity. In the present study, we measured membrane interaction of the prefibrillar and native states for three variants: the Y31 isoform studied previously, the wild-type protein and the G4R mutant; the latter is observed in progressive myoclonus epilepsy of type 1. In addition to using critical pressure and surface plasmon resonance, we assessed membrane permeabilization by calcein release and electrophysiological measurements. It was demonstrated for the first time that wild-type stefin B and the Y31 isoform are able to form pores in planar lipid bilayers, whereas G4R destroys the bilayer by a non pore-forming process. Similarities to other amyloidogenic proteins and the possible physiological implications of our findings are discussed.

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#17920138   2008/01/24 Save this To Up

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo.

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.

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#17701471   2007/08/16 Save this To Up

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

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#17287256   2007/03/29 Save this To Up

Cytosolic activation of cathepsins mediates parvovirus H-1-induced killing of cisplatin and TRAIL-resistant glioma cells.

Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the reduction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers.

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#17208452   2007/01/30 Save this To Up

Functional expression of recombinant human stefin A in mammalian and bacterial cells.

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.

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#16831429   2006/07/17 Save this To Up

Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases.

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the K(i) values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.

1265 related Products with: Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases.

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#16155205   2005/09/30 Save this To Up

In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1.

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.

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#14691222   2003/12/23 Save this To Up

Differences in aggregation properties of three site-specific mutants of recombinant human stefin B.

We describe expression, purification, and characterization of three site-specific mutants of recombinant human stefin B: H75W, P36G, and P79S. The far- and near-UV CD spectra have shown that they have similar secondary and tertiary structures to the parent protein. The elution on gel-filtration suggests that recombinant human stefin B and the P36G variant are predominantly monomers, whereas the P79S variant is a dimer. ANS dye binding, reflecting exposed hydrophobic patches, is highest for the P36G variant, both at pH 5 and 3. ANS dye binding also is increased for stefin B and the other two variants at pH 3. Under the chosen conditions the highest tendency to form amyloid fibrils has been shown for the recombinant human stefin B. The P79S variant demonstrates a longer lag phase and a lower rate of fibril formation, while the P36G variant is most prone to amorphous aggregation. This was demonstrated by ThT fluorescence as a function of time and by transmission electron microscopy.

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