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1.
Anal Chem ; 87(12): 6025-31, 2015 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-25965274

RESUMEN

Fourier-transform infrared (FTIR) spectroscopic imaging is a widely used method for studying the chemistry of proteins, lipids, and DNA in biological systems without the need for additional tagging or labeling. This technique can be especially powerful for spatially resolved, temporal studies of dynamic changes such as in vivo protein folding in cell culture models. However, FTIR imaging experiments have typically been limited to dry samples as a result of the significant spectral overlap between water and the protein Amide I band centered at 1650 cm(-1). Here, we demonstrate a method to rapidly obtain high quality FTIR spectral images at submicron pixel resolution in vivo over a duration of 18 h and longer through the development and use of a custom-built, demountable, microfluidic-incubator and a FTIR microscope coupled to a focal plane array (FPA) detector and a synchrotron light source. The combined system maximizes ease of use by allowing a user to perform standard cell culture techniques and experimental manipulation outside of the microfluidic-incubator, where assembly can be done just before the start of experimentation. The microfluidic-incubator provides an optimal path length of 6-8 µm and a submillimeter working distance in order to obtain FTIR images with 0.54-0.77 µm pixel resolution. In addition, we demonstrate a novel method for the correction of spectral distortions caused by varying concentrations of water over a subconfluent field of cells. Lastly, we use the microfluidic-incubator and time-lapsed FTIR imaging to determine the misfolding pathway of mutant copper-zinc superoxide dismutase (SOD1), the protein known to be a cause of familial amyotrophic lateral sclerosis (FALS).


Asunto(s)
Superóxido Dismutasa/química , Esclerosis Amiotrófica Lateral/enzimología , Animales , Células CHO , Supervivencia Celular , Células Cultivadas , Cricetulus , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factores de Tiempo
2.
PLoS One ; 14(4): e0215106, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30958859

RESUMEN

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/normas , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Cricetinae , Cricetulus , Humanos , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
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