Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris

Sarafotoxins are peptides isolated from the Atractaspisw snake venom. with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer, in the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris. and secreted to the culture medium, Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5 X 10(-10) M and 5 x 10(-11) M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlation of temperature induced conformation change with optimum catalytic activity in the recombinant G/11 xylanase A from Bacillus subtilis strain 168 (1A1)

The 1.7 angstrom resolution crystal structure of recombinant family G/11 beta-1,4-xylanase (rXynA) from Bacillus subtilis 1A1 shows a jellyroll fold in which two curved P-sheets form the active-site and substrate-binding cleft. The onset of thermal denaturation of rXynA occurs at 328 K, in excellent agreement with the optimum catalytic temperature. Molecular dynamics simulations at temperatures of 298-328 K demonstrate that below the optimum temperature the thumb loop and palm domain adopt a closed conformation. However, at 328 K these two domains separate facilitating substrate access to the active-site pocket, thereby accounting for the optimum catalytic temperature of the rXynA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

INFLUENCE OF DIETARY-PROTEIN INTAKE AND RECOMBINANT HUMAN SOMATOTROPIN ADMINISTRATION ON GROWTH AND BODY-COMPOSITION OF JUVENILE TAMBACU (A PIARACTUS-MESOPOTAMICUS X COLOSSOMA-MACROPOMUM CROSS)

This experiment was undertaken to study the interaction between level of dietary protein and recombinant human somatotropin (rhGH) administration on performance and body composition of juvenile tambacu (a crossbred Brazilian fish). A total of 72 juvenile tambacu, initially weighing and measuring (mean +/- s.e.m.) 23 +/- 2 g and 9 +/- 0.5 cm, respectively, were randomly divided into 18 groups of 4 fish each. Water temperature was 28 degrees C. Triplicate groups received one of two levels of dietary protein (15 and 30% as fed basis) and one of 3 doses of rhGH (0, 2 and 4 mu g/g) via intraperitoneal injection twice a week for 6 weeks, using a randomized complete block design. Somatotropin was noted to stimulate linear and body weight gain. The higher protein level supported increased growth in weight and length, but there was no interaction between protein level and rhGH dose for either parameter. Protein efficiency ratio and percentage protein deposited showed higher values on diets containing 15% protein. Somatotropin treatment did not significantly affect body composition, but there was a trend towards improved protein retention and reduced carcass lipid. In conclusion, the results of this experiment suggest that rhGH is able to stimulate linear gain in tambacu.

Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain

Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.

Effects of dominant follicle aspiration and treatment with recombinant bovine somatotropin (BST) on ovarian follicular development in Nelore (Bos indicus) heifers

Follicle ablation has been recognized as an efficient method of follicular wave synchronization. Treatment with recombinant bovine somatotropin (BST) has been shown to enhance follicular development in <(Bos taurus)under bar>. This experiment assessed the effects of these treatments in Nelore (<(B. indicus)under bar>) heifers. Eight cycling Nelore heifers were randomly assigned to 3 different treatments. on Day 2 of a synchronized cycle (Day 0 = day of ovulation), heifers assigned to Treatments 1 and 2 received 2 mL of saline, whereas heifers assigned to Treatment 3 received 320 mg of BST. on Day 5, the first-wave dominant follicle was ablated by ultrasoundguided transvaginal aspiration in heifers in Treatments 2 and 3, and all heifers received an injection of prostaglandin on Day 11. Aspiration of the dominant follicle advanced and synchronized (P < 0.05) the day of second-wave emergence (6.9 +/- 0.1 vs. 8.4 +/- 0.4) and the day of the pre-wave FSH peak (6.0 +/- 0.0 vs. 6.9 +/- 0.4), and increased FSH peak concentrations (381 +/- 21 vs. 292 +/- 30; pg/mL; P < 0.01). Recombinant bovine somatotropin treatment caused a two-fold increase in plasma insulin-like growth factor-I (IGF-I) concentrations (P < 0.001) and resulted in a 36% increase in the number of small follicles (<5 mm; P < 0.001) compared with saline-treated heifers. In summary, in agreement with previous reports on <(B. taurus)under bar>, dominant follicle aspiration synchronized ovarian follicular development, and BST treatment increased peripheral concentrations of IGF-I in Nelore heifers. Recombinant bovine somatotropin also increased the number of small follicles, but this response appeared to be inferior to that reported for <(B. taurus)under bar>. (C) 2000 by Elsevier B.V.

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier B.V. All rights reserved.

Effects of flunixin meglumine, recombinant bovine somatotropin and/or human chorionic gonadotropin on pregnancy rates in Nelore cows

The objective was to compare pharmacological strategies aiming to inhibit prostaglandin F2 alpha (PGF(2 alpha)) synthesis (flunixin meglumine; FM), stimulate growth of the conceptus (recombinant bovine somatotropin; bST) and progesterone (P(4)) synthesis (human chorionic gonadotropin; hCG), as well as their combinations, regarding their ability to improve pregnancy rates in beef cattle. Lactating Nelore cows (N = 975), 35 to 70 days postpartum, were synchronized and inseminated by timed artificial insemination (TAT) on Day 0. on Day 7, cattle were allocated into eight groups and received one of the following treatments: saline (S) on Days 7 and 16 (Group Control); S on Day 7 and FM on Day 16 (Group FM); bST on Day 7 and S on Day 16 (Group bST); bST on Day 7 and FM on Day 16 (Group bST + FM); hCG on Day 7 and S on Day 16 (Group hCG); hCG on Day 7 and FM on Day 16 (Group hCG + FM); bST and hCG on Day 7 and S on Day 16 (Group bST + hCG), or bST and hCG on Day 7 and FM on Day 16 (Group bST + hCG + FM). The aforementioned treatments were administered at the following doses: 2.2 mg/kg FM (Banamine (R); Intervet Schering-Plough, Cotia, SP, Brazil), 500 mg bST (Boostin (R); Intervet Schering-Plough), and 2500 IU hCG (Chorulon (R); Intervet Schering-Plough). Pregnancy diagnosis was performed 40 days after TAI by transrectal ultrasonography. Pregnancy rates were not significantly different among treatments. However, there was a main effect of hCG treatment to increase pregnancy rates (63.0 vs. 55.4%; P = 0.001). Concentrations of P(4) did not differ significantly among groups on Day 7 or on Day 16. However, consistent with the higher pregnancy rates, hCG increased P(4) concentrations on Day 16 (10.6 vs. 9.6 ng/mL, respectively; P = 0.05). We concluded that hCG treatment 7 days after TAI improved pregnancy rates of lactating Nelore cows, possibly via a mechanism leading to induction of higher P(4) concentrations, or by reducing the luteolytic stimulus during maternal recognition of pregnancy. (C) 2011 Elsevier B.V. All rights reserved.