RESUMO
Elucidating the intricate interactions between viral pathogens and host cellular machinery during infection is paramount for understanding pathogenic mechanisms and identifying potential therapeutic targets. The RNA modification N6-methyladenosine (m6A) has emerged as a significant factor influencing the trajectory of viral infections. Hence, the precise and quantitative mapping of m6A modifications in both host and viral RNA is pivotal to understanding its role during viral infection. With the rapid advancement of sequencing technologies, scientists are able to detect m6A modifications with various quantitative, high-resolution, transcriptome approaches. These technological strides have reignited research interest in m6A, underscoring its significance and prompting a deeper investigation into its dynamics during viral infections. This review provides a comprehensive overview of the historical evolution of m6A epitranscriptome sequencing technologies, highlights the latest developments in transcriptome-wide m6A mapping, and emphasizes the innovative technologies for detecting m6A modification. We further discuss the implications of these technologies for future research into the role of m6A in viral infections.
Assuntos
Adenosina/análogos & derivados , RNA , Viroses , Humanos , RNA/genética , TranscriptomaRESUMO
RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size-exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size-exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two-gradient size-exclusion chromatography refolding processes, the refolded rhNTA was purified by ion-exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS-PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg.
Assuntos
Fibrinolíticos/química , Renaturação Proteica , Proteínas Recombinantes/química , Ativador de Plasminogênio Tecidual/química , Arginina/química , Cromatografia , Escherichia coli/genética , Fibrinolíticos/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Compostos de Sulfidrila/análise , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Ureia/químicaRESUMO
Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme.