Cell-free expression of isotopically labeled HCV p7

The p7 protein sequence from HCV strain H77 genotype 1a was synthesized by DNA2.0. The gene was cloned directly into a pIVEX2.4d (Roche Applied Science) using NdeI/XhoI restriction sites. Cell-free expression was performed according to Soranzo et al.^{18 (link)}. Here, mixtures of either hydrogenated or deuterated amino acids (4.08 mg/mL) were used to express isotopically labelled proteins. Expressions reactions were performed at 30 °C for 9 hrs for NR and 6 hrs for electrophysiology measurements.

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Soranzo T., Martin D.K., Lenormand J.L, & Watkins E.B. (2017). Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers. Scientific Reports, 7, 3399.

Publication 2017

pIVEX2.4d

Amino acids Cell Dna2 Gene Genotype Protein sequence Proteins Strain

Corresponding Organization :

Other organizations : Translational Innovation in Medicine and Complexity, Institut Laue-Langevin

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Variable analysis

independent variables

Amino acid mixture used for protein expression (hydrogenated or deuterated)

dependent variables

Protein expression
Electrophysiology measurements

control variables

Protein sequence from HCV strain H77 genotype 1a
PIVEX2.4d (Roche Applied Science) plasmid with NdeI/XhoI restriction sites
Cell-free expression protocol as described in Soranzo et al. (2015)
Expression temperature (30 °C)
Expression duration (9 hrs for NR, 6 hrs for electrophysiology)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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