RESUMO
Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.
Assuntos
Anti-Helmínticos , Bacillus thuringiensis , Nematoides , Animais , Anti-Helmínticos/uso terapêutico , Proteínas de Bactérias , Citosol , Cavalos , Humanos , Projetos PilotoRESUMO
The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.
Assuntos
Evolução Molecular Direcionada/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , Periplasma/genética , Isomerases de Dissulfetos de Proteínas/genética , Trastuzumab/genética , Sítios de Ligação , Clonagem Molecular , Colífagos/genética , Colífagos/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inteínas/genética , Metiltransferases/metabolismo , Modelos Moleculares , Periplasma/metabolismo , Periplasma/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isomerases de Dissulfetos de Proteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trastuzumab/química , Trastuzumab/metabolismoRESUMO
Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.
