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[DESCRIPTION]
<p>A protein B18R is encoded by Western Reserve (WR) strain of <em>Vaccinia virus</em>. B18R works as a decoy receptor for the type I interferons (IFNs), attaches to cell surfaces, binds IFNs without prominent species-specificity and prevents an establishment of the antiviral state. At a same time B18R has no effect on cell viability itself. Thereunder B18R has become an essential aid in cell technologies which use RNA-mediated gene delivery. Despite an established high demand for this protein in cell technologies and also attractive prospects for use in mammalian expression systems to maintain RNA-vectors, little information is available on obtaining of B18R. In majority of works utilizing it, this protein has not been produced in a pure form. Rather, conditioned media (CM) are collected from cell cultures infected with <em>Vaccinia</em><em> virus</em> or transfected with a B18R mRNA. The CM containing secretory-expressed B18R are used without an isolation of B18R. As the production in cell cultures is expensive, a development of a bacterial expression system is warranted. Here is described the bacterial expression and a method for refolding of recombinant B18R. Multi-milligram quantities of recombinant B18R are easily produced and the product is biologically active despite bacterially-expressed B18R is not glycosylated. It has been successfully utilized to support persistence of a RNA vector derived from a genome of a RNA-virus in cell cultures capable of the antiviral state.</p>
<p>A synthetic gene B18R which encodes the aminoacid residues His20-Glu351 from the Genbank entry D01019 is used in this work. The B18R gene sequence is codon-optimized for the optimal expression in E.coli. A sequence of an expression plasmid pET28-B18R(HisTag) is deposited to the Genbank MG356786. The expression product has a hexahistidine tag at the N-terminus which is used for a metal-affinity purification at one step of the protocol.</p>

[BEFORE_START]
<p>Required solutions:</p>
<p>Lysis buffer: 20% sucrose, 20 mM HEPES pH 7.5, 5 mM MgCl<sub>2</sub>, 0.1% Triton-X100. Store at RT.</p>
<p>TE 1<sup>X</sup> for preparing of solutions: 10 mM Tris-HCl pH 7.2, 1 mM EDTA. Store at RT.</p>
<p>Lysozyme stock: 100 mg/ml lysozyme (solution in 1<sup>x</sup> TE). Store frozen at -20°C.</p>
<p>DNAse I stock: 1 mg/ml DNAse I (solution in 1<sup>x</sup> TE). Store frozen at -20°C.</p>
<p>RNase stock: 10 mg/ml RNase A (solution in 1<sup>x</sup> TE). Store frozen at -20°C.</p>
<p>PMSF stock: 0.2 M phenylmethylsulfonyl fluoride (PMSF) (solution in isopropanol). Store frozen at -20°C.</p>
<p>Wash buffer with sodium deoxycholate (SDC): 100 mM Tris pH 8.0, 2 mM EDTA, 0.5% SDC. Store at RT.</p>
<p>Buffer TE 1<sup>X</sup> to wash inclusion bodies: 100 mM Tris pH 8.0, 2 mM EDTA. Store at RT.</p>
<p>Solubilization buffer: 50 mM Tris pH 10, 2% sodium lauroyl sarcosinate (SLS). Store at RT.</p>
<p>CuSO<sub>4</sub> stock: 0.2 M cupric sulfate (CuSO<sub>4</sub>) solution in water. Store at RT.</p>
<p>Buffer A for metal-affinity chromatography (IMAC) containing 6M urea: 20 mM Na<sub>2</sub>HPO<sub>4</sub> pH 7.4, 500 mM NaCl, 20 mM imidazole, 6M urea. Store at RT.</p>
<p>Buffer B for IMAC+6M urea: 20 mM Na<sub>2</sub>HPO<sub>4</sub> pH 7.4, 500 mM NaCl, 500 mM imidazole, 6M urea. Store at RT.</p>
<p>Phosphate-buffered saline (PBS): prepared from tablets (Amresco Cat. E404). Store at RT.</p>

[GUIDELINES]
<p>All chemical reagents mentioned in the protocol were purchased from Sigma-Aldrich and are of ACS or Reagent quality. A vector pET28c (Novagen) was used to construct an expression plasmid pET28-B18R(HisTag) which sequence is deposited to the Genbank entry MG356786. Cells of a <em>E.coli</em> strain BL21(DE3) were transformed with the plasmid pET28/B18R(HisTag) to produce the expression strain used in this protocol.</p>

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