Deleterious effects of Schinus terebinthifolius Raddi seed flour on cowpea weevil, Callosobruchus maculatus (F.), larval development.

Schinus terebinthifolius, Raddi, has been extensively studied due to its anti-inflammatory and antibiotic properties. S. terebinthifolius was also toxic to some insects, however little has been explored about the nature of its insecticide compounds or the toxicity of this plant to insect species. In this work, we investigate the toxicity of S. terebinthifolius seed flour against the insect C. maculatus. S. terebinthifolius seed flour interfered with the post hatch development of the C. maculatus larvae, decreasing larval survival, mass and length. Using DEAE-cellulose chromatography, five protein fractions were isolated, a non-retained fraction (NRF) and four retained fractions, eluted with 0.25, 0.5, 0.7 and 1.0 M NaCl. Proteins with varying molecular masses were observed in all fractions. The majority protein bands were identified by mass spectrometry analysis and among the main identified proteins are 11S globulins (such glycinin), lipoxygenase, chitinases, 7S globulins (vicilins, canavalin and ß conglycinin), annexin, catalase and sucrose binding protein. All DEAE-protein fractions were toxic to the insect, interfering with the post hatch larval development and survival. Decreases greater than 90% were observed in the larval mass and length at 20 days after oviposition (DAO) for larvae raised on diet containing 0.5% of some fractions. Alterations in the level of proteins, glucose and in the activity of the enzymes lipases and cysteine proteases were also detected in these larvae. Our results show that seeds of S. terebinthifolius have an arsenal of toxic proteins with potential for the control of the insect C. maculatus.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation.

Genes encoding proteins 'toxic' to recombinant host are difficult for cloning/expression and recombinant clones are unstable. Even tightly controlled inducible T7-lac, PBAD, PL, PR promoters are not totally silent in an uninduced state and thus not adequate for highly toxic proteins. An innovative approach to engineering and expression of the gene, encoding bacterial alkaline phosphatase (BAP) is proposed. The native precursor enzyme contains a signal peptide at the N-terminus and is secreted to the Escherichia coli (E. coli) periplasm. The signal peptide is then removed that allows oxidation and formation of active dimers. To decrease toxicity of the bap gene, its secretion leader coding section was replaced with a N-terminal His6-tag. The gene was expressed in E. coli in a PBAD vector, resulting in the accumulation of soluble His6-BAP in the cytoplasm. The His6-BAP was neutral to the cells, as no maturation was possible in the reducing cytoplasm. The purified homogenous protein was further reactivated in a redox buffer containing the protein structure stabilizing cofactors. The His6-BAP exhibited high activity. A dephosphorylation protocol for all types of DNA termini was developed.The method appears well suited for the industrial production of BAP and can be applied to other problematic proteins.• Efficient toxic gene expression • Novel approach to toxic gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme • Scaled-up production of ultrapure BAP • Improved protocol for all types of DNA termini dephosphorylation.

Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01.

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.

Chemical Modifications of Vicilins Interfere with Chitin-Binding Affinity and Toxicity to Callosobruchus maculatus (Coleoptera: Chrysomelidae) Insect: A Combined In Vitro and In Silico Analysis.

Vicilins are related to cowpea seed resistance toward Callosobruchus maculatus due to their ability to bind to chitinous structures lining larval midgut. However, this binding mechanism is not fully understood. Here, we identified chitin binding sites and investigated how in vitro and in silico chemical modifications interfere with vicilin chitin binding and insect toxicity. In vitro assays showed that unmodified vicilin strongly binds to chitin matrices, mainly with acetylated chitin. Chemical modifications of specific amino acids (tryptophan, lysine, tyrosine), as well as glutaraldehyde cross-linking, decreased the evaluated parameters. In silico analyses identified at least one chitin binding site in vicilin monomer, the region between Arg208 and Lys216, which bears the sequence REGIRELMK and forms an α helix, exposed in the 3D structure. In silico modifications of Lys223 (acetylated at its terminal nitrogen) and Trp316 (iodinated to 7-iodine-L-tryptophan or oxidized to ß-oxy-indolylalanine) decreased vicilin chitin binding affinity. Glucose, sucrose, and N-acetylglucosamine also interfered with vicilin chitin binding affinity.

Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin.

BACKGROUND: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. OBJECTIVE: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. METHODS: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. RESULTS: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 - 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. CONCLUSION: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.

Clinical Use of Toxic Proteins and Peptides from Tian Hua Fen and Scorpion Venom.

Traditional Chinese Medicine (TCM) has been practiced in China for thousands of years. As a complementary and alternative treatment, herbal medicines that are frequently used in the TCM are the most accepted in the Western world. However, animal materials, which are equally important in the TCM practice, are not well-known in other countries. On the other hand, the Chinese doctors had documented the toxic profiles of hundreds of animals and plants thousand years ago. Furthermore, they saw the potential benefits of these materials and used their toxic properties to treat a wide variety of diseases, such as heavy pain and cancer. Since the 50s of the last century, efforts of the Chinese government and societies to modernize TCM have achieved tremendous scientific results in both laboratory and clinic. A number of toxic proteins have been isolated and their functions identified. Although most of the literature was written in Chinese, this review provide a summary, in English, regarding our knowledge of the clinical use of the toxic proteins isolated from a plant, Tian Hua Fen, and an animal, scorpion, both of which are famous toxic prescriptions in TCM.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

Current trends in Bt crops and their fate on associated microbial community dynamics: a review.

Cry protein expressing insect-resistant trait is mostly deployed to control major devastating pests and minimize reliance on the conventional pesticides. However, the ethical and environmental issues are the major constraints in their acceptance, and consequently, the cultivation of genetically modified (GM) crops has invited intense debate. Since root exudates of Bacillus thuringiensis (Bt) crops harbor the insecticidal protein, there is a growing concern about the release and accumulation of soil-adsorbed Cry proteins and their impact on non-target microorganisms and soil microbial processes. This review pertains to reports from the laboratory studies and field trials to assess the Bt toxin proteins in soil microbes and the processes determining the soil quality in conjunction with the existing hypothesis and molecular approaches to elucidate the risk posed by the GM crops. Ecological perturbations hinder the risk aspect of soil microbiota in response to GM crops. Therefore, extensive research based on in vivo and interpretation of results using high-throughput techniques such as NGS on risk assessment are imperative to evaluate the impact of Bt crops to resolve the controversy related to their commercialization. But more studies are needed on the risk associated with stacked traits. Such studies would strengthen our knowledge about the plant-microbe interactions.

Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells.

BACKGROUND: Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. RESULTS: Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. CONCLUSION: Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins.

Enterolobin induces rat paw oedema independently of PAF-acether

The potential participation of PAF-acether (PAF) on the paw oedema triggered by enterolobin was investigated. Intraplantar injections of enterolobin )5-20 µg/paw) yielded a dose response curve for edema which appeared after 30 min, peaked in the interval between 2-4 h and faded after 24h. The pre-treatment with BN 52021, but not with other PAF antagonists such as PCA 4248 or WEB 2086, significantly blocked enterolobin-induced oedema. To clarify better the discrepant results obtained with the PAF antagonists, desensitization to PAF was performed. The oedema triggered by enterolobin was not modified in paf desensitized animals. It was concluded that the paw inflammation induced by enterolobin does not require PAF mechanism.