Oxidative refolding chromatography: folding of the scorpion toxin Cn5

Altamirano, Myriam M.; García, Carlos Bosch; Possani, Lourival D.; Fersht, Alan R.

doi:10.1038/6192

Cited by 119 publications

(68 citation statements)

References 44 publications

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“…Both recombinant proteins CD1d and ␤ 2 m were expressed in the Escherichia coli strain BL21 and purified from inclusion bodies as described (25). Oxidative refolding chromatography was applied for in vitro refolding of CD1d-␤ 2 m complexes in the presence of either ␣-GalCer (Kirin Brewery, Gunma, Japan), ␣-Mannosylceramide (Kirin Brewery), ␤-GalCer (Fluka) or ganglioside GM1 (G-7641 Sigma-Aldrich) lipid ligands, following a protocol previously described (30)(31)(32). Soluble protein fractions were concentrated to a volume of 3 ml and biotinylated directly as described (25).…”

Section: Methodsmentioning

confidence: 99%

“…Here we describe the successful use of the technique of in vitro oxidative refolding chromatography (30,31) to engineer human CD1d-␤ 2 m-glycolipid tetramers from fully denatured and reduced recombinant CD1d proteins. This technique combines the benefits of the GroEL minichaperone, which prevents hydrophobic interactions of early folding intermediates with disulphide and prolyl isomerases, which catalyze native disulphide bond formation and proline isomerization, respectively.…”

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confidence: 99%

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Human CD1d–glycolipid tetramers generated byin vitrooxidative refolding chromatography

Karadimitris

Gadola

Altamirano

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

170

154

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CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipidspecific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone-and foldase-assisted refolding of denatured CD1d molecules and ␤2-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-␣-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Human CD1d–glycolipid tetramers generated byin vitrooxidative refolding chromatography

Karadimitris

Gadola

Altamirano

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

170

154

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“…3A, 2005 a difficult task in the field. 45 Thus, the results reported here, open the possibility of preparing much more material for future research related to the structure and function of these toxins. Figure 5A and B shows that CllErg1 ( -KTx1.5) potently suppressed the HERG current amplitude.…”

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confidence: 98%

Amino acid sequence determination and chemical synthesis of CllErg1 (gamma-KTx1.5), a K+ channel blocker peptide isolated from the scorpion Centruroides limpidus limpidus

Coronas

Balderas

Pardo-López

et al. 2005

J. Braz. Chem. Soc.

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Uma nova toxina denominada CllErg1 (nomenclatura sistemática -KTx1.5) foi purificada do veneno do escorpião Centruroides limpidus limpidus e a sua seqüência de amino ácidos foi determinada. Ela tem 42 resíduos de amino ácidos entrecruzados por quatro pontes de disulfetos e bloqueia especificamente um canal de potássio da família eter-a-go-go (ERG). O peptídeo completo foi quimicamente sintetizado e adequadamente enrolado, mostrando que é capaz de bloquear o canal-ERG humano (HERG) com uma afinidade idêntica ao peptídeo nativo. A toxina CllErg1 sintética pode ser produzida em quantidades suficientes para compensar a sua baixa concentração encontrada no veneno natural. Isto prepara o caminho para conduzir estudos enfocando a identificação dos padrões estruturais do HERG que são críticos para o funcionamento adequado do canal iônico. Adicionalmente, outro peptídeo análogo CllErg2 (nome sistemático -KTx4.1) foi purificado e a sua seqüência completa de amino ácidos foi determinada. Este contém 43 resíduos de amino ácidos, mantidos de forma compacta por quatro pontes de disulfetos.A novel toxin named CllErg1 (systematic nomenclature -KTx1.5) was purified from the venom of the scorpion Centruroides limpidus limpidus and its amino acid sequence was determined. It has 42 amino-acid residues cross-linked by four disulfide bridges and blocks specifically a potassium channel of the family ether-a-go-go (ERG). The full peptide was chemically synthesized and properly folded, showing that it blocks the human ERG-channels (HERG) with identical affinity to that of the native peptide. Synthetic CllErg1 can be produced in quantities enough to compensate its low concentration in the natural venom. It paves the way to conduct studies aimed at the identification of the structural motifs of HERG critical for proper channel function. Additionally, another analogous peptide CllErg2 (systematic name -KTx4.1) was purified and had its full amino acid sequence determined. It contained 43 amino acid residues, maintained closely packed by four disulfide bridges. 1 The electric potential of these cells is maintained by active pumps and ion-channels.2 One of these channels is the inward rectifier potassium channel (IKr).3 The IKr voltage gated potassium ion current contributes to the repolarization phase of cardiac action potential and the non-specific blockade of cardiac IKr current is a significant contributor to fatal cardiac arrhythmias associated with prolongation of the QT interval. 4 The human ether-a-go-go related gene (h-erg) encodes a potassium channel (HERG) that is responsible for the current Ikr. 5,6 Mutations in HERG predispose patients to long QT syndrome and sudden cardiac arrest. 5,7,8 Blockage of the channel by commonly used medications, such as class III antiarrhythmics, antihistaminics and antipsychotics can also lead to long QT. In most circumstances arrhythmias associated with long QT and sudden death is a result of a combination of genetic, environmental and pharmacological factors. 9 The HERG channels, are also expressed...

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“…However, the monomeric substrate binding domain, minichaperone, alone can assist the refolding of several proteins (9)(10)(11)(12)(13). The chaperone activity of the minichaperone comes from the weak binding of the folding intermediate of the substrate proteins (K d ϭ 200 M, ref.…”

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confidence: 99%

The substrate binding domain of DnaK facilitates slow protein refolding

Tanaka

Nakao

Wadai

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of ␤-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active ␤-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of ␤-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.T he molecular chaperone DnaK, a member of the HSP70 family in Escherichia coli, is involved in the folding and assembly of newly synthesized polypeptide chains and in preventing the aggregation of stress-denatured proteins. The chaperone activity of DnaK is controlled by ATP (1, 2). When ADP is bound to the N-terminal ATPase domain (residues 1-384), the adjacent C-terminal substrate binding domain (residues 385-638) tightly binds the substrate. ATP bound to the ATPase domain induces a change in the substrate binding domain to a low affinity state for the substrate. The 26-kDa substrate binding domain of DnaK, which contains the ␤-sheet domain and the transitional helix of the substrate binding domain, harbors the substrate binding cleft (residues 393-507) and a 14-kDa domain from residues 508 to 638, of which the first 100 residues are ␣-helical and appear to act as a lid covering the substrate binding cleft (3). Substrate binding occurs by a dynamic mechanism in a two-layered closing device involving independent action of an ␣-helical lid and an arch. Mayer et al. (4) suggested that the ADP-and ATP-bound states of DnaK differ in the frequency of conformational changes that occur in the ␣-helical lid and the ␤-domain that cause an opening of the substrate binding cavity. Previous studies showed that ATP and the cochaperone DnaJ, which stimulate the ATPase activity, are necessary to facilitate the refolding of proteins from chemically denatured states (5, 6).The activity of GroEL, another major chaperone in E. coli, is also controlled by the hydrolysis of ATP and a cochaperone (7,8). However, the monomeric substrate binding domain, minichaperone, alone can assist the refolding of several proteins The chaperone activity of DnaK384-638 was examined for the ability to refold ␤-galactosidase and luciferase from chemically denatured states. These proteins have been used a...

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Oxidative refolding chromatography: folding of the scorpion toxin Cn5

Cited by 119 publications

References 44 publications

Human CD1d–glycolipid tetramers generated byin vitrooxidative refolding chromatography

Human CD1d–glycolipid tetramers generated byin vitrooxidative refolding chromatography

Amino acid sequence determination and chemical synthesis of CllErg1 (gamma-KTx1.5), a K+ channel blocker peptide isolated from the scorpion Centruroides limpidus limpidus

The substrate binding domain of DnaK facilitates slow protein refolding

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