4 research outputs found
Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin
Background: Gardnerella vaginalis is a bacterial vaginosis
(BV)-associated vaginal bacterium that produces the toxin vaginolysin
(VLY). VLY is a pore-forming toxin that is suggested to be the main
virulence factor of G. vaginalis. The high recurrence rate of BV and
the emergence of antibiotic-resistant bacterial species demonstrate the
need for the development of recombinant antibodies as novel therapeutic
agents for disease treatment. Single-chain variable fragments (scFvs)
generated against VLY exhibited reduced efficacy to neutralize VLY
activity compared to the respective full-length antibodies. To improve
the properties of scFvs, monospecific dimeric scFvs were generated by
the genetic fusion of two anti-VLY scFv molecules connected by an
alpha-helix-forming peptide linker. Results: N-terminal
hexahistidine-tagged dimeric scFvs were constructed and produced in
Escherichia coli and purified using metal chelate affinity
chromatography. Inhibition of VLY-mediated human erythrocyte lysis by
dimeric and monomeric scFvs was detected by in vitro hemolytic assay.
The circulating half-life of purified scFvs in the blood plasma of mice
was determined by ELISA. Dimeric anti-VLY scFvs showed higher
neutralizing potency and extended circulating half-life than parental
monomeric scFv. Conclusions: The protein obtained by the genetic fusion
of two anti-VLY scFvs into a dimeric molecule exhibited improved
properties in comparison with monomeric scFv. This new recombinant
antibody might implement new possibilities for the prophylaxis and
treatment of the diseases caused by the bacteria G. vaginalis
Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis
Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis
Influenza Virus Ribonucleoprotein Complexes Gain Preferential Access to Cellular Export Machinery through Chromatin Targeting
In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration
Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity
Background: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker. Methods: The recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model. Results: The novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70–140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF