Recombinant ZIKV NS5 RdRp Purification

ZIKV NS5 RdRp polymerase was cloned at pETTRX by the LIC method and expressed and purified according to the protocol described in [92 (link)]. Briefly, NS5 RdRp polymerase was expressed in ZYM 5052 auto-induction medium and purified in four steps: (i) a HisTrap HP 5.0 mL with a Ni Sepharose resin (GE Healthcare, Sao Carlos, Brazil); (ii) a buffer exchanged by dialysis and a concomitant TEV protease cleavage from 6His-TRX-tag; (iii) an inverse HisTrap HP 5.0 mL to separate protein from 6His-TRX-tag and (iv) a size-exclusion chromatography at a XK 16/60 Superdex 75 column (GE Healthcare, Sao Carlos, Brazil).

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Ramos P.R., Mottin M., Lima C.S., Assis L.R., de Oliveira K.Z., Mesquita N.C., Cassani N.M., Santos I.A., Borba J.V., Fiaia Costa V.A., Neves B.J., Guido R.V., Oliva G., Jardim A.C., Regasini L.O, & Andrade C.H. (2022). Natural Compounds as Non-Nucleoside Inhibitors of Zika Virus Polymerase through Integration of In Silico and In Vitro Approaches. Pharmaceuticals, 15(12), 1493.

Publication 2022

Ni Sepharose resin XK 16/60 Superdex 75 column

Buffer Cleavage Dialysis Protein Resin Sepharose Size exclusion chromatography Tev protease Zikv

Corresponding Organization : Universidade Estadual Paulista (Unesp)

Other organizations : Universidade Federal de São Carlos, Universidade de São Paulo, Universidade Federal de Uberlândia

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Variable analysis

independent variables

Expression of ZIKV NS5 RdRp polymerase

dependent variables

Purification of ZIKV NS5 RdRp polymerase

control variables

Expression of ZIKV NS5 RdRp polymerase in ZYM 5052 auto-induction medium
Purification of ZIKV NS5 RdRp polymerase in four steps: (i) HisTrap HP 5.0 mL with a Ni Sepharose resin, (ii) buffer exchange by dialysis and TEV protease cleavage, (iii) inverse HisTrap HP 5.0 mL, and (iv) size-exclusion chromatography at a XK 16/60 Superdex 75 column

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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