Cofactor requirement of HpyAV restriction endonuclease.

BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

Purification of cell culture-derived influenza virus A/Puerto Rico/8/34 by membrane-based immobilized metal affinity chromatography.

The presented study focuses on the feasibility of immobilized metal affinity chromatography for purification of Madin Darby canine kidney cell culture-derived influenza virus particles. Therefore, influenza virus A/Puerto Rico/8/34 was screened for adsorption to different transition metal ions attached to iminodiacetic acid. Subsequently, capturing of the same virus strain using zinc-modified iminodiacetic acid membrane adsorbers was characterized regarding viral recoveries, host cell nucleic acid and total protein depletion as well as zinc-ion-leaching. In addition, the effect of the imidazole proton pump on virus stability was studied based on the hemagglutination activity. During adsorption in the presence of 1M sodium chloride the majority of virus particles were recovered in the product (64% hemagglutination activity). Host cell nucleic acid and total protein content were reduced to approximately 7 and 26%, respectively. This inexpensive and rapid method was applied reproducibly for influenza virus A/Puerto Rico/8/34 preparations on the laboratory scale. However, preliminary results with other virus strains indicated clearly a strong strain dependency for viral adsorption.

Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles.

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine sulfate.

Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent.

Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.

Impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses.

The study aims on affinity matrix selection for a cell culture derived influenza virus capture step in downstream processing. Euonymus europaeus lectin (EEL) was used as an affinity ligand. Human influenza A/Puerto Rico/8/34 (H1N1) virus produced in MDCK cells was chosen as a model strain. The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine sulphate and heparinized agarose. The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.

Lectin-affinity chromatography for downstream processing of MDCK cell culture derived human influenza A viruses.

The presented study aims on the development of a capture step for the purification of cell culture derived influenza viruses using lectin affinity chromatography. Human influenza A/Puerto Rico/8/34 virus produced in Madin Darby canine kidney cells have been chosen as a model. The influenza A virus envelop possesses two viral glycoproteins: hemagglutinin and neuraminidase. Oligosaccharides of theses glycoproteins can be targeted as affinity ligands using specific lectins. First, lectins have been screened via lectin blots and spin columns. Adequate lectins have been chosen based on published glycan structures of hemagglutinin. The most specific binding was achieved via the galactose specific Erythrina cristagalli and Euonymus europaeus lectins. Second, the chromatographic separations characteristics of these lectins have been further determined via FPLC. These experiments revealed that the rate of hemagglutinin glycan binding to the ligands was higher with the E. europaeus compared to the E. cristagalli lectin. Third, viral recoveries in addition to the total protein and host cell DNA have been balanced in a series of E. europaeus lectin chromatography runs. The total protein and dsDNA content in the product fraction of the affinity chromatography was reduced from the starting conditions to 21% and 0.1%, respectively. The average viral recovery in the product fraction was 97%. SDS-PAGE analysis indicated that the majority of the eluted proteins were of viral origin. The reproducibility and column stability was confirmed in up to 25 runs applying six different virus product batches.