High-levelexpression of functional recombinant human coagulation factor VII in insect cells

Abstract: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. Keywords: Baculovirus; γ-carboxylase; Coagulation FVII; Factor VII; Insect cel

MOESM2 of The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion

Additional file 2. Western blot analysis of hPML isoforms overexpressed in PML-KO MEFs

MOESM3 of The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion

Additional file 3. Sequences of primer ODNs used in this study

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

Background. Factor VII is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. Construction, expression and purification of recombinant FVII fused to poly histidin tag through gateway technology were aimed in this study. Methods. To construct entry clone, blunt-end FVII cDNA and subsequent PCR product isolated from HepG2 cell line was TOPO cloned into pENTR TOPO vector. To construct expression clone, LR recombination reaction was carried out between entry clone and destination vector, pDEST26. CHO c ells were transfected with 1 g of DNA of PDEST26 FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant factor VII were established. The expression of recombinant FVII was confirmed by RT-PCR and ELISA. Culture medium containing his-FVII was added to the nickel-nitrilotriacetic acid resin c olumn and bound protein was eluted. The purified protein was detected by SDS-PAGE and western blot analysis. Biological activity of the recombinant factor VII was determined by prothrombin time assay using factor FVII-depleted plasma. Results. The results showed that human recombinant FVII successfully was cloned and accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but any expression was not detected in the CHO cells containing empty vector. A protein of about 52KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed by using this rFVII. Conclusion. As we are a ware, this is the first report of expression of recombinant FVII fused with his-tag through gateway technology. The next steps including large scale expression, purification, activation and stabilization are underwa y

HIV-1 capsids from B27/B57+ elite controllers escape Mx2 but are targeted by TRIM5α, leading to the induction of an antiviral state.

Elite controllers (ECs) are a rare subset of HIV-1 slow progressors characterized by prolonged viremia suppression. HLA alleles B27 and B57 promote the cytotoxic T lymphocyte (CTL)-mediated depletion of infected cells in ECs, leading to the emergence of escape mutations in the viral capsid (CA). Whether those mutations modulate CA detection by innate sensors and effectors is poorly known. Here, we investigated the targeting of CA from B27/B57+ individuals by cytosolic antiviral factors Mx2 and TRIM5α. Toward that aim, we constructed chimeric HIV-1 vectors using CA isolated from B27/B57+ or control subjects. HIV-1 vectors containing B27/B57+-specific CA had increased sensitivity to TRIM5α but not to Mx2. Following exposure to those vectors, cells showed increased resistance against both TRIM5α-sensitive and -insensitive HIV-1 strains. Induction of the antiviral state did not require productive infection by the TRIM5α-sensitive virus, as shown using chemically inactivated virions. Depletion experiments revealed that TAK1 and Ubc13 were essential to the TRIM5α-dependent antiviral state. Accordingly, induction of the antiviral state was accompanied by the activation of NF-κB and AP-1 in THP-1 cells. Secretion of IFN-I was involved in the antiviral state in THP-1 cells, as shown using a receptor blocking antibody. This work identifies innate activation pathways that are likely to play a role in the natural resistance to HIV-1 progression in ECs

Pre-screening of isolated clones by specific PCR.

<p>Following 20 days of growth, 161 isolated HEK293T clones were screened for HDR-edited TRIM5 gene by PCR using a primer specific for the mutated <i>TRIM5</i>. 14 clones that passed this pre-screen step are indicated by their names. MWM, molecular weight marker.</p

Design of the gRNA and donor ssODN for the HDR-mediated editing of <i>TRIM5</i>.

<p>(A) <i>TRIM5</i> localization on chromosome 11 (top), and Arg332-Arg335 localization in exon 8 of the gene (bottom). (B) Top panel: position of the three gRNAs (gRNA1, 9 and 19) designed to target the Arg332-Arg335 region. The two arginine codons are underlined and in bold. Bottom panel: Surveyor assay following the transfection of HEK293T cells with CRISPR-Cas9 plasmids expressing one of the three gRNAs. WT DNA from untransfected cells was used as a control. (C) HDR donor DNA mutagenesis strategy. 8 substitutions were present, including three nonsilent substitutions to mutate Arg332 and Arg335 into Gly (green), one silent mutation to disrupt the PAM sequence (pink), and four silent mutations in the sequence targeted by gRNA1 (orange). The HaeIII restriction site created as a result of one of the silent substitutions is indicated, as is the position of the primer used in specific PCR screening.</p

Identification of HDR-edited clones.

<p>(A) Mutation-specific PCR was performed on 13 clones showing a positive signal in the pre-screen. Untransfected HEK293T cells were used as a control. M, molecular weight marker. (B) Non-specific PCR of the targeted region followed by HaeIII digestion. The expected sizes of the digested PCR products are shown on the right. The full-length gels are available on the FigShare public repository (see “Availability of data” section).</p

Restriction by exogenous or endogenous TRIM5α is inefficient in HEK293T cells.

<p>(A) HEK293T and THP-1 cells were retrovirally transduced with WT huTRIM5α, R332G-R335G huTRIM5α or with the “empty” vector as indicated. Untransduced cells were eliminated by hygromycin treatment, and the cell populations were then challenged with increasing amounts of the HIV-1_NL-GFP vector. The percentage of cells expressing GFP was then determined by FACS.(B) HEK293T Jurkat cells were infected with increasing amounts of N-MLV_GFP and B-MLV_GFP. The amounts of virus used are expressed as multiplicities of infection (MOI) as calculated in the non-restrictive CRFK cells. The percentage of infected cells was determined by FACS. For N-MLV in Jurkat cells, only the 3 virus doses that yielded detectable infections are shown. (C) HEK293T cells were treated with IFN-α, IFN-β or IFN-ω for 16 h prior to a single-dose infection with N-MLV_GFP and B-MLV_GFP, designed to yield 10–20% infected cells in the absence of IFN-I. The percentage of infected cells was determined by FACS.</p