Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering

O projeto de profissionalização docente no contexto da reforma educacional iniciada nos anos 1990

Este artigo focaliza a reforma da formação inicial de professores da educação básica, em implementação pelo Estado brasileiro desde os anos de 1990. Os elementos centrais dessa reforma evidenciam que o processo de profissionalização toma por base conceitos e práticas que têm origem no campo do trabalho. A noção de competências ocupa lugar central e implementa uma nova lógica educativa, subordinando a esta o currículo e a organização das instituições de formação, objetivando construir um novo tipo de professor, com capacidades subjetivas consoantes àquelas demandadas pelo mercado e pelas novas formas de sociabilidade exclusiva que caracterizam as sociedades capitalistas contemporâneas. O dispositivo legal mais recente - a Portaria nº. 1.403, de 9 de junho de 2003, que pretendeu instituir o Sistema Nacional de Certificação e Formação Continuada de Professores, deu continuidade a esse processo de reforma referenciado em uma perspectiva técnico-instrumental, mas foi recentemente sustado pelo novo dirigente do Ministério de Educação, sob forte pressão do movimento dos educadores. As propostas pelas quais luta o movimento docente nas últimas décadas - formação de qualidade, incentivo às faculdades e centros de educação das universidades como espaços privilegiados de formação de professores, construção da profissionalização e da autonomia e do desenvolvimento intelectual do docente, precisam ser recuperadas neste momento, para que se transformem em efetivas políticas públicas

A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations:implications for peptide-based analgesics

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor