Purification of Viral Replication Enzymes

NS5 polymerase, NS2B-NS3 protease, and NS3 helicase were cloned at pETTRX- or pETSUMO by LIC method, expressed and purified according to the protocols described by Silva and coworkers (2019)^{91 (link)}, Lima and coworkers (2021)^{92 (link)} and Godoy and coworkers (2017)^{93 (link)}. Briefly, the proteins were expressed in ZYM 5052 auto-induction medium or Luria Bertani (LB) medium and purified in four steps: (i) a HisTrap HP 5.0 mL with a Ni Sepharose resin (GE Healthcare); (ii) a buffer exchanged by dialysis and a concomitantly TEV protease cleavage from 6His-TRX-tag or 6His-SUMO-tag; (iii) an inverse HisTrap HP 5.0 mL to separate protein from 6His-TRX-tag and 6His-SUMO-tag; and (iv) a size-exclusion chromatography at an XK 16/60 Superdex 75 column (GE Healthcare).

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Mottin M., de Paula Sousa B.K., de Moraes Roso Mesquita N.C., de Oliveira K.I., Noske G.D., Sartori G.R., de Oliveira Albuquerque A., Urbina F., Puhl A.C., Moreira-Filho J.T., Souza G.E., Guido R.V., Muratov E., Neves B.J., da Silva J.H., Clark A.E., Siqueira-Neto J.L., Perryman A.L., Oliva G., Ekins S, & Andrade C.H. (2022). Discovery of new Zika protease and polymerase inhibitors through the open science collaboration project OpenZika. Journal of chemical information and modeling, 62(24), 6825-6843.

Publication 2022

Ni Sepharose resin XK 16/60 Superdex 75 column

Corresponding Organization : Universidade Federal de Goiás

Other organizations : Universidade de Brasília, Universidade de São Paulo, Fundação Oswaldo Cruz, Collaborations Pharmaceuticals (United States), University of North Carolina at Chapel Hill, Universidade Federal da Paraíba, University of Montana, University of California, San Diego, Rutgers, The State University of New Jersey

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Variable analysis

independent variables

Expression system (ZYM 5052 auto-induction medium or Luria Bertani (LB) medium)
Purification method (4 steps: HisTrap HP 5.0 mL with Ni Sepharose resin, buffer exchange and TEV protease cleavage, inverse HisTrap HP 5.0 mL, size-exclusion chromatography)

dependent variables

Protein expression and purification yield

control variables

Proteins cloned (NS5 polymerase, NS2B-NS3 protease, and NS3 helicase)
Cloning method (LIC method)
Expression vectors (pETTRX- or pETSUMO)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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