Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis)

<p>Abstract</p> <p>Background</p> <p>The cotton boll weevil (<it>Anthonomus grandis</it>) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. <it>In vitro </it>directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of <it>Bacillus thuringiensis</it>.</p> <p>Results</p> <p>Bioassays carried out with <it>A. grandis </it>larvae revealed that the LC₅₀of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability.</p> <p>Conclusions</p> <p>The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control <it>A. grandis</it>.</p

Produção de memórias falsas com listas de associados : análise do efeito do nível de processamento e da natureza da prova de memória

As memórias falsas têm sido amplamente estudadas com base num procedimento experimental designado paradigma DRM (Deese/Roediger/McDermott). Esse paradigma promove a criação de ilusões de memória a partir da apresentação de listas de palavras associadas a um item que não consta da lista. Uma das linhas de investigação com o paradigma DRM visa identificar o momento da criação das falsas memórias e explicar os mecanismos que estão na sua origem. Neste artigo, pretendemos fazer uma revisão da investigação sobre o efeito do nível de processamento e da natureza da tarefa de memória na facilitação ou inibição da produção de memórias falsas com listas de associados semânticos.False memories have been widely studied using an experimental procedure called DRM paradigm (Deese/Roediger/McDermott). This paradigm produces memory illusions due to the presentation of lists of words associated to a critical nonpresented word. One line of research on this topic aims at identifying the moment when the false memories are created and the explanation of the mechanisms underling false memories. In this paper we present a review about the effect of level-of-processing and the nature of memory task for the boost or inhibition of false memories created by means of lists of semantic associates.Le paradigme DRM (Deese/Roediger/McDermott) est un des plus connus et plus robustes parmi les études des faux mémoires dans le contexte du laboratoire. Ce paradigme permet la création d illusions de mémoire à partir des mots sémantiquement associés à un item qui n a pas été présenté. Au milieu des investigations basées sur le paradigme DRM il y a des études dont l objectif est d identifier e d´expliquer les mécanismes qui sont à l origine de la production des faux mémoires. Plus spécifiquement, on a pour but de faire une révision de la recherche sur l effet du niveau de codification et de la nature des tâches de mémoire sur la facilitation ou l´inhibition de la production de faux mémoires à partir des mots sémantiquement associés.Los falsos recuerdos han sido muy estudiados mediante la aplicación del paradigma DRM (Deese/Roediger/McDermott). El paradigma permite producir ilusiones de memoria tras la presentación de listas de palabras asociadas a una palabra que no se incluye en la lista. Una de las líneas de investigación que utilizan el paradigma DRM busca identificar el preciso momento de la creación de falsos recuerdos y explicar los mecanismos que originan ese efecto. El objetivo de este artículo es hacer una revisión de la investigación sobre el efecto de los niveles de procesamiento y la naturaleza de la tarea de memoria en la facilitación y inhibición de la producción de falsos recuerdos con listas de asociados semánticos.Fundação para a Ciência e a Tecnologia (FCT)Centro de Investigação em Psicologia da Universidade do Minho (CIPsi

The Use of Phage-Displayed Peptide Libraries to Develop Tumor-Targeting Drugs

Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Combinatorial biological libraries such as bacteriophage-displayed peptide libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding peptides can be affinity selected from complex mixtures of billions of displayed peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several peptides derived from phage-displayed peptide library screenings have been developed into therapeutics in current clinical trials, which validates peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited

Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering

Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4+ T cells and 17% in CD34+ hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20–30% in CD4+ T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering

Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome

<div><p>Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.</p></div

Serine recombinase structure.

<p>Important regions within each recombinase monomer (red and blue) are labeled. DNA shown in grey sticks. Native DNA-binding domains can be replaced with customizable zinc-finger or TAL effector domains to generate chimeric recombinases (PDB ID: 1GDT) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139123#pone.0139123.ref065" target="_blank">65</a>].</p

Recombination efficiency of selected Bin and Tn21 catalytic domain variants.

<p>The activity of selected <b>(A)</b> Bin and <b>(B)</b> Tn21 catalytic domains was evaluated against a panel of cognate and non-cognate target sites. Red highlighted variants were selected for further analysis. Recombination was determined by split gene assembly.</p