The phospholipases A(2) (PLA(2), E.C. 3.1.1.4, phosphatide sn2 acylhydrolases) are the major components of the venom of several snakes. They are responsible for several important pharmacological effects observed in op...
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The phospholipases A(2) (PLA(2), E.C. 3.1.1.4, phosphatide sn2 acylhydrolases) are the major components of the venom of several snakes. They are responsible for several important pharmacological effects observed in ophidian incidents. PLA(2) piratoxin II from Bothrops pirajai has been crystallized by the vapour-diffusion technique. X-ray diffraction data have been collected to 2.04 Angstrom resolution (90.2% complete, R-merge = 0.070). The space group is P2(1) and the cell parameters are a = 46.19, b = 60.36, c = 58.74 Angstrom and beta = 96.05 degrees. re structure has been solved by molecular replacement using the crystallographic structure of PLA(2) from Bothrops asper (PDB code 1CLP) as a search model.
Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic c...
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Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P2(1) with cell dimensions a = 79.1, b = 89.2, c = 145.4 Angstrom and beta = 92.8 degrees, and diffract to at least 2.6 Angstrom. The orthorhombic crystals belong to space group P2(1)2(1)2 with a = 89.4, b = 155.9, c = 79.5 Angstrom, and diffract to 2.9 Angstrom. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centring. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to the understanding of protein thermostability.
Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent amino-transferase, has been crystallized in space groups P2(1) and C2. Both crystal forms were obtained at pH 7.3 ...
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Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent amino-transferase, has been crystallized in space groups P2(1) and C2. Both crystal forms were obtained at pH 7.3 with 21% polyethylene glycol and 10% 2-propanol as precipitants. The cell dimensions were a = 130, b = 57.5, c = 117 Angstrom, beta = 110 degrees for the C2 crystals, and a = 58.4, b = 55.6, c = 121 Angstrom, beta = 96.9 degrees for the P21 crystals, which diffract to at least 2.6 and 2.0 Angstrom resolution, respectively.
Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial in...
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Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL. This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL). This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8616-8620). Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E. coli in inclusion bodies and must be denatured and refolded to become active. This requires correct folding, formation of native disulfide bonds, and the association of different domains. All these steps are strongly dependent on the renaturation conditions used. Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as L-arginine. Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution. This approach should be useful for the production of active forms of other recombinant proteins.
Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondr...
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Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae. The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S. cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S. cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed. The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix. Absorption spectra and electron transfer activity of the isolated fusion protein established that the [2Fe-2S] center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.
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