Functional characterisation of an engineered multidomain human P450 E1 by molecular Lego

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (K (M)=1.84 +/- 0.09 mM and k (cat) of 2.98 +/- 0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (K (M)=0.65 +/- 0.08 mM and k (cat) of 0.95 +/- 0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Properties of a unique mutant of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus that exhibits a partial many polyhedra and few polyhedra phenotype on extended serial passaging in suspension cell cultures

Serial passaging of wild-type Helicoverpa armigera, single-nucleocapsid (HaSNPV) in H. zea (HzAMI) illsect Cell Cultures results ill rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. Oil serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type Virus. However, subsequent passaging of ppC19 resulted in a steel) decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus Lit high passage numbers. Genomic deoxyribonueleic Licid profiling of the latter suggested that defective interfering particles (DIPS) were implicated in this phenomenon and represented another Undesirable mutation during serial passaging of HaSNPV Hence, a strategy to isolate HaSNPV Clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPS, could prove commerically invaluable.

Towards the preparative and large-scale precision manafacture of virus-like particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery, but their potential has yet to be fully realized. This is because existing laboratory processes, when scaled, do not easily give a compositionally and architecturally consistent product. Research suggests that new process routes might ultimately be based on chemical processing by self-assembly, involving the precision manufacture of precursor capsomeres followed by in vitro VLP self-assembly and scale-up to required levels. A synergistic interaction of biomolecular design and bioprocess engineering (i.e. biomolecular engineering) is required if these alternative process routes and, thus, the promise of new VLP products, are to be realized.

In vitro production of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus

Naturally occurring insect viruses are a promising means of intentionally causing disease in insects but they do not compete successfully with synthetic chemicals in the commercial marketplace. Furthermore, their use for pest control is still restricted. One factor preventing the development of baculoviruses as effective biopesticides is concern over the production issue. In vitro instability during propagation of these viruses in suspension cells is the major limitation to the in vitro production ofbaculoviruses in cell cultures. In this study, an isolated baculovirus (HaSNPV) was cultivated using serial passaging in a suspension cell culture. The results show a reduction in the occlusion body production during six passages, due to the passage effect. However the purification of an HaSNPV clone suggested better stability. A simple method used in this work for the serial passaging of this virus is discussed.

Eukaryotic expression:Developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. © International Union of Crystallography, 2006.

Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508

Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [3H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.

Recombinant expression of Aryl Hydrocarbon Receptor for quantitative ligand-binding analysis

Recombinant expression of the Aryl Hydrocarbon Receptor (AhR) yields small amounts of ligand- binding competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding competent AhR was sensitive to the protein concentration of Sf9 extract, and co-expression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20- fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding competent AhR, and sufficient AhR for quantitative analysis of ligand-binding.

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Análise do impacto da deleção do gene p94 (ac134) de autographa californica multiple nucleopolyhedovirus

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós Graduação em Biologia Molecular, 2015.

Estudio molecular en dos hermanas afectadas por ictiosis congénita autosómica recesiva : descripción de una nueva mutación causal en TGM1

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos.

Controle de Helicoverpa armigera (Lepidoptera: Noctuidae) em soja com inseticidas químicos e biológicos.

RESUMO: O objetivo deste trabalho foi avaliar o desempenho de inseticidas autorizados emergencialmente para o controle de Helicoverpa armigera (Lepidoptera: Noctuidae) em soja. Sete inseticidas foram pulverizados em campo e, após 24 horas, folhas do ponteiro foram coletadas e oferecidas para lagartas de 2o instar em laboratório. Lagartas do 4o instar receberam a última folha trifoliolada que se encontrava completamente expandida no momento da pulverização. Outro grupo foi exposto a folhas coletadas a partir de 72 horas da pulverização. Em campo, seis inseticidas foram pulverizados e, em seguida, as plantas foram infestadas com lagartas de 2o e 3o instar. No primeiro estudo, flubendiamida, clorantraniliprole, clorfenapir, indoxacarbe e metoxifenozida causaram 100% de mortalidade do 4o instar aos oito dias após o início da exposição, enquanto baculovírus e Bacillus thuringiensis (Bt) propiciaram mortalidade de 60-75%, que evoluiu para 88?90% ao final da fase de pupa. Para o 2o instar, apenas flubendiamida e clorantraniliprole proporcionaram mortalidade de 100%. Flubendiamida, clorantraniliprole e clorfenapir apresentaram o menor tempo letal para o 4o instar, e flubendiamida e clorantraniliprole, para o 2o instar. Após 72 horas da pulverização, o desempenho dos inseticidas foi insatisfatório. Em campo, houve eficiência satisfatória de flubendiamida, espinosade, baculovírus e Bt sobre lagartas de 2o e 3o instar. ABSTRACT:The objective of this work was to evaluate the performance of insecticides authorized on an emergency basis to control of Helicoverpa armigera (Lepidoptera: Noctuidae) in soybean. Seven insecticides were sprayed on the field and, 24 hours after that, soybean pointer leaves were collected and offered to 2nd instar larvae in the laboratory. Fourth instar larvae received the last trifoliate leaf that was fully expanded at the time of spraying. Another larvae group was exposed to leaves collected from 72 hours onwards after spraying. In the field, six insecticides were sprayed, and then the plants were infested with 2nd and 3rd instar larvae. In the first study, flubendiamide, chlorantraniliprole, chlorfenapyr, indoxacarb, and methoxyfenozide caused 100% mortality of the 4th instar, eight days after the beginning of exposure, while baculovirus and Bacillus thuringiensis (Bt) caused 60?75% mortality, which reached 88?90% at the end of the pupal stage. For 2nd instar larvae, only flubendiamide and chlorantraniliprole caused 100% mortality. Flubendiamide, chlorantraniliprole, and chlorfenapyr showed the lowest lethal time for the 4th instar, and flubendiamide and chlorantraniliprole for the 2nd instar. Seventy-two hours after spraying, the performance of insecticides was not satisfactory. In the field, there was satisfactory efficiency of flubendiamide, spinosad, baculovirus, and Bt on 2nd and 3rd instar larvae.