Isolation of inclusion bodies from vegetative Clostridium perfringens: partial purification of a 47 kDa inclusion protein

J.S. GARCIA-ALVARADO, R.G. LABBÉ AND M.A. RODRIGUEZ. 1992. A refractile inclusion body produced by vegetative cells of Clostridium perfringens at temperatures above 40C was isolated and partially characterized. The inclusion was composed of protein and could be solubilized by sodium dodecyl sulphate plus either dithiothreitol or β-mercaptoethanol. The solubilized inclusion showed no antigenic relationship with Cl. perfringens enterotoxin. One major band with an apparent MW of 47 kDa was demonstrated after polyacrylamide gel electrophoresis of the solubilized inclusion. Both enterotoxin-positive and enterotoxin-negative strains produced the inclusion body. No effect on the morphology of several eucaryotic cell lines was observed when solubilized or intact inclusion was added to the cell cultures.     

Isolation of an inclusion body from sporulating, enterotoxin-positive Clostridium perfringens

Abstract Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens were isolated. Sporulating cells were lysed by sonication in the presence of protease inhibitors. IB were isolated by centrifugation in linear gradients of sucrose, sodium bromide or sodium diatrizoate and banded at buoyant densities of 1.33–1.36 g/cm³, 1.30–1.34 g/cm³ and 1.33 g/cm³, respectively. Isolated IB were treated with detergent to remove attached cell membrane. They ranged in size from 0.5–1.4 μm long and from 0.2–0.5 μm wide. They were found to be serologically related to purified enterotoxin.     

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens.

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).     

Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts.     

Improved method for purification of enterotoxin from Clostridium perfringens type A.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.     

Isolation of inclusion bodies from rabbit lung parenchyma

The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.     

Identification, Isolation and characterization of a novel azoreductase from Clostridium perfringens

Azo dyes are used widely in the textile, pharmaceutical, cosmetic and food industries as colorants and are often sources of environmental pollution. There are many microorganisms that are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced thereby a concern for human health. The field of research on azoreductases is growing, but there is very little information available on azoreductases from strict anaerobic bacteria. In this study, the azoreductase gene was identified in Clostridium perfringens, a pathogen that is commonly found in the human intestinal tract. C. perfringens shows high azoreductase activity, especially in the presence of the common dye Direct Blue 15. A gene that encodes for a flavoprotein was isolated and expressed in Escherichia coli, and further purified and tested for azoreductase activity. The azoreductase (known as AzoC) was characterized by enzymatic reaction assays using different dyes. AzoC activity was highest in the presence of two cofactors, NADH and FAD. A strong cofactor effect was shown with some dyes, as dye reduction occurred without the presence of the AzoC (cofactors alone). AzoC was shown to perform best at a pH of 9, at room temperature, and in an anaerobic environment. Enzyme kinetics studies suggested that the association between enzyme and substrate is strong. Our results show that AzoC from C. perfringens has azoreductase activity.     

Isolation and purification of phospholipase C from Clostridium perfringens and antibodies against it using polymeric biospecific adsorbents

A new polymeric biospecific adsorbent intended for isolation of Clostridium perfringens phospholipase C (PLC) and PLC-specific antibodies is discussed. It was obtained by radical copolymerization of acrylamide, methylenbisacrylamide, and the acryl acid chloranhydride-acylated substrate of PLC (chicken yolk lecithovitellin). Maximal adsorption of PLC was observed in the presence of the enzyme activator, and the highest amount of PLC was eluted in case of its minimal adsorption. The "residual" activity of PLC on the adsorbent was used to isolate homologous antibodies from the anti-perfringens serum. PLC and PLC-specific antibodies were serologically pure in the agar precipitation test with the anti-perfringens serum and the primary PLC concentrate, respectively; the antibodies gave only one zone in PAAG-electrophoresis.     

Modeling of protein refolding from inclusion bodies

Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies （IBs）, which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble pro- teins following dilution refoiding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical proper- ties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high effi- cacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight （R2= 0.98, P = 0.000, F-test）. Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refold- ing developed here could be used to predict the refold- ing yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refoiding from IBs according to their intrinsic properties.     

Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies.

The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro. In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins. The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction. The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA. Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function.     

Improved method for purification of enterotoxin from Clostridium perfringens type A

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.     

Isolation of cell-free bacterial inclusion bodies

Background  

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.     

Isolation of a cDNA encoding a 70 kDa heat-shock cognate protein expressed in vegetative tissues ofArabidopsis thaliana

A cDNA encoding a 70 kDa heat shock cognate protein (hsc70) was isolated fromArabidopsis thaliana by using a rat hsc70 cDNA as probe. Sequence analysis demonstrated the conservation of functional domains and important amino acid residues among hsc70s in plants and animals. The expression of this gene was stress-inducible, and was found at a substantial level during normal growth in root, stem, leaf and flower tissues, but not in siliques. Multiple copies of this gene exist in theArabidopsis genome.     

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P. brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Pb18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P. brasiliensis.