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1.
MAbs ; 7(3): 505-15, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25759214

RESUMEN

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.


Asunto(s)
Secuencia de Aminoácidos , Anticuerpos Monoclonales , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Humanos , Estabilidad Proteica
2.
Proc Natl Acad Sci U S A ; 106(30): 12317-22, 2009 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-19590013

RESUMEN

Since its discovery, the light-gated cation channel Channelrhodopsin-2 (ChR2) has proven to be a long-sought tool for the noninvasive, light-activated control of neural cells in culture and in living animals. Although ChR2 is widely used in neurobiological applications, little is known about its molecular mechanism. In this work, the unitary conductance of ChR2 was determined for different cations, for example 40 fS at 200 mM NaCl and -60 mV, using noise analysis. The kinetics of the ion channel obtained by noise analysis is in excellent agreement with the photocurrent kinetics obtained by voltage-clamp and time-resolved spectroscopy. The inward rectification of the channel could be explained by the single channel parameters. ChR2 represents an ion channel with a 7 transmembrane helix motif, even though the sequence homology of its essential amino acids to those of the light-driven H(+) pump bacteriorhodopsin (bR) is high. Here, we also show that when ChR2 is expressed in electrofused giant HEK293 cells or reconstituted on planar lipid membranes, it can indeed act as an outwardly driven H(+) pump, demonstrating that ChR2 is bifunctional, and in-line with other microbial rhodopsins, a H(+) pump but with a leak that shows ion channel properties.


Asunto(s)
Proteínas Portadoras/fisiología , Canales Iónicos/fisiología , Bombas de Protones/fisiología , Proteínas Portadoras/genética , Línea Celular , Relación Dosis-Respuesta a Droga , Guanidina/farmacología , Humanos , Canales Iónicos/genética , Cinética , Luz , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp , Bombas de Protones/genética , Cloruro de Sodio/farmacología , Temperatura , Transfección
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