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1.
Nat Commun ; 8(1): 1500, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29138389

RESUMO

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.


Assuntos
Bacteriófago M13/genética , Proteínas do Capsídeo/genética , Biblioteca de Peptídeos , Peptídeos/genética , Sequência de Aminoácidos , Bacteriófago M13/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Modelos Genéticos , Peptídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
2.
Molecules ; 16(5): 3675-700, 2011 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-21540796

RESUMO

Since the initial description of phage display technology for the generation of human antibodies, a variety of selection methods has been developed. The most critical parameter for all in vitro-based approaches is the quality of the antibody library. Concurrent evolution of the libraries has allowed display and selection technologies to reveal their full potential. They come in different flavors, from naïve to fully synthetic and differ in terms of size, quality, method of preparation, framework and CDR composition. Early on, the focus has mainly been on affinities and thus on library size and diversity. Subsequently, the increased awareness of developability and cost of goods as important success factors has spurred efforts to generate libraries with improved biophysical properties and favorable production characteristics. More recently a major focus on reduction of unwanted side effects through reduced immunogenicity and improved overall biophysical behavior has led to a re-evaluation of library design.


Assuntos
Anticorpos , Biblioteca de Peptídeos , Animais , Humanos , Modelos Biológicos
3.
Methods ; 34(2): 215-24, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15312674

RESUMO

The intracellular expression of highly specific antibody fragments ("intrabodies") in eukaryotes has a great potential in functional genomics and therapeutics. However, since the intracellular reducing environment prevents formation of the conserved intrachain disulfide bonds, most antibodies do not fold properly and are therefore inactive inside cells. The few antibodies that have been found to function in an intracellular environment and that have been characterized for their biophysical properties have generally shown a high degree of stability and solubility. Thus, for intracellular expression and application, very stable antibody frameworks are needed that can correctly fold even in the absence of disulfide bonds and that do not aggregate. Here, we present and discuss a novel method, named "Quality Control," which allows selection of stable and soluble antibody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibody fragments (scFvs) fused to a selectable marker that can control gene expression and cell growth. The activity of such a selectable marker fused to various scFvs that have been biophysically characterized correlated with the solubility and stability of the scFv moieties. This antigen-independent intrabody selection system was applied to screen scFv libraries for identifying stable and soluble frameworks, which subsequently served as acceptor backbones to construct intrabody libraries by randomization of hypervariable loops.


Assuntos
Anticorpos/química , Antígenos/imunologia , Líquido Intracelular/química , Anticorpos/genética , Antígenos/química , Antígenos/genética , Linhagem Celular , Regulação da Expressão Gênica , Terapia Genética/métodos , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/imunologia
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