RESUMO
Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium.
Assuntos
Elastina/biossíntese , Peptídeos/metabolismo , Pichia/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Estabilidade de Medicamentos , Elastina/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Metanol/farmacologia , Dados de Sequência Molecular , Peptídeos/genética , Pichia/efeitos dos fármacos , Biossíntese de ProteínasRESUMO
Elastin-like polypeptides (ELPs) functionalized with azide or alkyne groups were produced biosynthetically and coupled via the Cu-catalyzed azide-alkyne cycloaddition to a variety of (bio)molecules.
Assuntos
Alcinos/química , Azidas/química , Elastina/química , Oligopeptídeos/química , Catálise , Cobre/química , Elastina/genética , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Oligopeptídeos/genética , Engenharia de ProteínasRESUMO
The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.
Assuntos
Azidas/química , Técnicas de Sonda Molecular , Proteínas/química , Enzimas Imobilizadas/química , Peroxidase do Rábano Silvestre/química , Proteínas Luminescentes/química , Lisina/química , Água , Proteína Vermelha FluorescenteRESUMO
N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from omega-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the NHS-ester moieties with amines at room temperature. Using a combination of analytical techniques (infrared reflection absorption spectroscopy (IRRAS), extensive X-ray photoelectron spectroscopy (XPS) in combination with density functional theory calculations of the XPS chemical shifts of the carbon atoms, atomic force microscopy (AFM), and static contact angle measurements), it was shown that the NHS-ester groups were attached fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with para-trifluoromethyl benzylamine and biotin hydrazide.
Assuntos
Silício/química , Succinimidas/química , Biotina/análogos & derivados , Biotina/química , Ésteres/química , Microscopia de Força Atômica , Estrutura Molecular , Fotoquímica , Análise Espectral , Propriedades de SuperfícieRESUMO
Polymersomes, composed of amphiphilic polystyrene-block-poly(acrylic acid) (PS-b-PAA), with the periphery being covered with azide groups, were used for further functionalization using "click" chemistry.