Accumulation of G-CSF in incubation media of HEK293 and BHK21 cultures transfected with a G-CSF-producing VEE replicon.

<p>Data for two producing cultures based on BHK-21 or HEK293 are shown. A presence of B18R in incubation media significantly increases yields of the product of recombinant expression (G-CSF) driven by the VEE replicon.</p

Analytical gel filtration of B18R and its complex with IFN-alpha 1.

<p>Panel a, lysozyme and BSA were run through a SEC column to obtain reference retention times. Retention times are shown above corresponding peaks. Panel b, B18R migrates during the SEC as a monomer (retention time 6.80 min). In a mixture of B18R with IFN-alpha a complex is formed which retention time (4.45 min) indicates that a stoichiometry of the complex is 1:1. Inset in panel b: fragment of an SDS-PAGE gel on which a material was loaded from the peaks at 4.45 min (lane 1) and 6.80 min (lane 2).</p

SDS-PAGE of samples produced during purification and refolding of B18R.

<p>Panel A. Lanes: 1, a solution of inclusion bodies in an SLS-containing buffer; 2, a solution after 20-hour incubation in a presence of CuSO₄ for oxidation; 3, a sample of B18R after precipitation to remove SLS, dissolved in buffer A with 6M urea; 4–8, fractions (1 ml) collected during elution of B18R from an IMAC column. M—protein Mw marker (Sigma Cat. C1992), molecular masses of the marker’s bands indicated. Panel B. Results of ion exchange chromatography. Two lanes beside a marker’s lane were loaded with 10 ug of purified B18R. Arrow points at a band of B18R (Mw 38.9 kDa).</p

Part of the expression plasmid with a synthetic gene B18R.

<p>The deduced translation of recombinant B18R is shown.</p

Recombinant <i>Vaccinia virus</i>-coded interferon inhibitor B18R: Expression, refolding and a use in a mammalian expression system with a RNA-vector

<div><p>B18R protein of <i>Vaccinia virus</i> binds to type I interferons and inhibits activation of interferon-mediated signal transduction. Cells which have unimpaired interferon signaling such as primary cell cultures or some industrially important cell lines are capable of development of an antiviral state. An establishment of the antiviral state limits replication of RNA-viruses and can suppress replication of RNA vectors. The interferon inhibitor B18R effectively prevents the establishment of the antiviral state. For this reason, B18R has become a ubiquitous component of protocols for epigenetic reprogramming which use transfections of RNA replicons or mRNA. Despite wide practical applicability, commercially available B18R is predominantly produced in cell cultures and little information has been published on a production and use of bacterially expressed B18R. Objectives of this study were to produce B18R in an <i>E</i>.<i>coli</i> expression system and to confirm the product’s biological activity by using it to maintain RNA-vectors in cell cultures capable of the antiviral state. The described method allows the expression and efficient refolding to obtain 10–100 mg of B18R from a small-scale culture and the production process is economically attractive compared to a use of an eukaryotic expression. To check for a presence of the biological activity of bacterially-expressed B18R the protein was used to support persistence of an autonomously replicating RNA-vector in a cell culture which is capable of the antiviral state. A RNA-containing virus, Venezuelan equine encephalitis virus (VEE) can serve as an efficient vector for heterologous expression in cell cultures, although its replication is sensitive to the effects of type I interferons which limit a range of cell lines for a use with this vector. The VEE replicon was utilized to direct an expression of recombinant human granulocyte colony stimulating factor (G-CSF). The producing replicon could persist in HEK293 cells for sufficiently long time only in presence of B18R, whereas addition of B18R not only allowed persistence of the replicon but also increased production from the replicon. A model product granulocyte colony stimulating factor accumulated to 35.5 μg/ml during a 7 day experiment. This work describes efficacious expression and refolding of the viral cytokine inhibitor and demonstrates a utility of bacterially-expressed B18R.</p></div

Genetic maps of the VEE genomes.

<p>Upper part: a VEE genetic map. Boxes indicate genes for individual functional proteins. Middle part: the replicon RepVEE-GFP. Positions of restriction sites used for an assembly of the replicon are indicated. Lower part: the replicon RepVEE-Pac-2A-SigP-GCSF. Designations: SP—subgenomic promoter; PAC—puromycin N-acetyltransferase; FMDV 2A - autoprotease 2A; SigP—signal peptide for a secretory expression; G-CSF—granulocyte colony stimulating factor.</p

B18R enables persistence of the VEE replicon in HEK293 culture.

<p>Time after transfections indicated in captions above panels. Puromycin was added to all cultures 24 hrs after the transfections.</p

GFP fluorescence in cultures transfected with the replicon RepVEE-GFP, 24 hrs after transfection.

<p>Panels: a, BHK-21, no B18R; b, BHK-21, with B18R; c, HEK293, no B18R; d, HEK293 with B18R.</p