Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

Engineering regulable Escherichia coli beta-galactosidases as biosensors for anti-HIV antibody detection in human sera.

The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.

Albumin purification from human placenta.

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin-purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross-flow ultrafiltration (CFUF) membrane; (iv) thermo-coagulation at 70 degrees C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion-exchange chromatography Q-Sepharose; (viii) hydrophobic-interaction chromatography with phenyl-Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A(403) of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.

Purification of catalase from human placenta.

The therapeutic use of an antioxidant complex containing superoxide dismutase and catalase has been proposed for the treatment of several diseases in which reactive oxygen species have an important role. Although superoxide dismutase for human use is commercially available, methods for the production of catalase for human use have not been described. An industrial process was developed for the purification of catalase for human use as a by-product of albumin production from human placenta, comprising two parts: (1) albumin and catalase co-purification steps, including blood extraction from ground placentas, precipitation of haemoglobin with ethanol/chloroform, concentration/diafiltration by tangential filtration and anionic chromatography, by which non-adsorbed catalase was separated from albumin; and (2) catalase purification steps after albumin separation, including a second anionic chromatography step and dye-affinity chromatography. This method provided a final recovery of 27% (70-100% in each step) with 670-fold purification of catalase (85% pure) and a specific activity of 49000 units/mg, which is higher than that of commercially available human catalase. This process permits the co-purification of catalase and albumin and can easily be scaled up.

Purification of fibroblast growth factor-2 from human placenta using tri(n-butyl)phosphate and sodium cholate.

Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.

Purification of superoxide dismutase from placental haemolisate blood: a simple and efficient method.

Superoxide dismutase functions as a scavenger of superoxide radical protecting living organisms. This enzyme has potential use as anti-inflammatory or anti-reperfusion injury drug. Here we present a simple and efficient SOD purification method from human placental blood. Superoxide dismutase from clarified haemolysed placental blood after chloroform and ethanol treatment was purified by DEAE-Sepharose, Phenyl-Sepharose chromatographies and cross flow ultrafiltration. The purified product is 98% pure by SDS-PAGE with 71% yield and specific activity of 2.8 x 10(5) U/mg protein.

Purification of basic fibroblast growth factor and alkaline phosphatase from human placenta.

Human placenta is an available hospital waste which is known to contain many valuable biochemicals that may be commercially exploited. Using placental tissue previously extracted for haemoderivatives, we purified basic fibroblast growth factor (bFGF), a soluble protein, and placental alkaline phosphatase (PALP), a membrane-linked protein, as a coupled process. bFGF purification comprises three steps: extraction and chromatographies on S-Sepharose and heparin-Sepharose. The final product includes a major 17 kDa and a minor 16 kDa component with a specific activity of 8.0 x 10(6) units/mg yielding 0.5-1.0 microgram/kg of placenta. PALP purification comprises four steps: acidic butan-1-ol extraction and chromatographies on Q-Sepharose, concanavalin A-Sepharose and Q-Sepharose. The purified PALP has a molecular mass of 70 kDa, a specific activity of 800 units/mg and yielded 50 micrograms/kg of placenta. The results show the possibility of purifying substances in placental haemolysed blood, soluble products from placental cellular mass and proteins from the cellular membrane in a one-stream process.

Structure and expression of genes coding for xylan-degrading enzymes of Bacillus pumilus.

The complete nucleotide sequence of the beta-xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617-bp open reading frame for beta-xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N-terminal region and the molecular mass 62607 Da) of the beta-xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine-Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG. The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3' end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197-201]. The results of the Northern hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5' and 3' ends of the xynA and xynB gene were mapped with nuclease S1. The '-10' regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the '-35' regions were different from all the known promoters for B. subtilis RNA polymerases.

Extracellular oligosaccharides and low-Mr polysaccharides containing (1----2)-beta-D-glucosidic linkages from strains of Xanthomonas, Escherichia coli and Klebsiella pneumoniae.

One strain each of Xanthomonas campestris and 'Xanthomonas phaseoli', three strains of 'Xanthomonas oryzae', and five strains each of Escherichia coli and Klebsiella pneumoniae were found to produce a mixture of (1----2)-beta-D-gluco-oligosaccharides and/or low-Mr (1----2)-beta-D-glucans in their culture media. The saccharides from the strains of Xanthomonas were all composed of unbranched, linear (1----2)-beta-D-glucosaccharides with degrees of polymerization (DPs) of 8 to about 20, and a cyclic (1----2)-beta-D-glucan (DP16) containing one (1----6)-linkage and one alpha-linkage. The saccharides produced by the five strains of E. coli and four strains of K. pneumoniae were glucans with branches at O-6, and DPs of 10 to 15, whereas one strain of K. pneumoniae produced unbranched linear (1----2)-beta-D-glucosaccharides with DPs of 6 to about 20.