RESUMEN
CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/genética , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Factores de Integración del Huésped/genética , Secuencia de Bases , Sitios de Unión , Proteínas Asociadas a CRISPR/inmunología , Sistemas CRISPR-Cas/inmunología , ADN Bacteriano/química , ADN Bacteriano/inmunología , Endodesoxirribonucleasas/inmunología , Endonucleasas/inmunología , Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Factores de Integración del Huésped/inmunología , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Señales de Clasificación de Proteína/genética , Estructura Secundaria de ProteínaRESUMEN
Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.