Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-7

Protein was eluted with 10 mM maltose, cleaved with Factor Xa, and purified by SEC. (A) Western blot of protein in (lane 2) amylose capture eluate, (lane 3) Factor Xa cleavage reaction, and (lane 4) pooled SEC fractions. The blot was probed with LASV mAb mix containing GP2-specific mAbs, then detected with an HRP-conjugated goat α-mouse IgG antibody. (Lane 1) Western blot XP molecular weight markers, with sizes (kDa) shown to the left of the panel. (B) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) amylose capture eluate, (lane 3) Factor Xa cleavage reaction, and (lane 4) SEC-purified GP2 generated from pooled GP2-containing fractions. (Lane 1) SeeBluePlus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of the panel. GP2, MBP, and GP2-MBP are indicated.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-3

Ins were incubated with LASV mAb mix, then detected with an HRP-conjugated goat α-mouse IgG antibody and TMB. For negative controls, proteins were incubated with irrelevant mouse IgG (MsIgG) or with an HRP-conjugated goat α-mouse IgG antibody, then detected as above.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-6

Protein was eluted with 10 mM maltose, cleaved with Factor Xa, and purified by SEC. (A) Western blot of protein in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, (lanes 5 and 6) SEC-purified GP1 generated from pooled GP1-containing fractions. The blot was probed with LASV mAb mix containing GP1-specific mAbs, then detected with an HRP-conjugated goat α-mouse IgG antibody. (Lane 1) Western blot XP standard molecular weight markers, with sizes (kDa) shown to the left of the panel. (B) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, and (lanes 5 and 6) purified GP1 generated from two sequential SEC runs. (Lane 1) SeeBluePlus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of the panel. GP1, MBP, and GP1-MBP are indicated.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-0

at the 3' end of the E gene, beyond the cleavage site for Factor Xa (IQGR). The LASV GP1 gene sequence comprised a.a. 59–259 in the native GPC, spanning the first a.a. beyond the known SPase cleavage site at position 58 to the junction between GP1 and GP2 domains, which is cleaved by the SKI-1/S1P protease at a.a. 259. The LASV GP2 gene sequence comprised a.a. 260–427, spanning the first a.a. of mature GP2 to the last a.a. before the predicted TM domain. The LASV NP gene sequence comprised the complete ORF of the gene, with the exception of the N-terminal Met. The 3' oligonucleotides used for amplification of each gene sequence were engineered to contain two terminator codons separated by a single nucleotide. All genes were cloned into vectors pMAL-p2x and pMAL-c2x for periplasmic and cytoplasmic expression of fusion proteins, respectively, in Rosetta 2(DE3) or gami 2 strains. The a.a. position of each LASV gene domain is noted, as are REN sites. Abbreviations include: MBP gene (E), MBP promoter (P), philamentous phage origin of replication (M13 ori), bacterial origin of replication (pBR322 ori), beta-lactamase gene (), terminator (rrnB), the LacZ alpha-complementation domain (LacZα), and the lacI repressor gene (lacI). The periplasmic secretory domain in pMAL-p2x is indicated by a black box on the 5' end of the E gene sequence.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-1

Protein was eluted with 10 mM maltose, cleaved with Factor Xa, and purified by SEC. (A) Western blot of protein in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, (lanes 5 and 6) SEC-purified GP1 generated from pooled GP1-containing fractions. The blot was probed with LASV mAb mix containing GP1-specific mAbs, then detected with an HRP-conjugated goat α-mouse IgG antibody. (Lane 1) Western blot XP standard molecular weight markers, with sizes (kDa) shown to the left of the panel. (B) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, and (lanes 5 and 6) purified GP1 generated from two sequential SEC runs. (Lane 1) SeeBluePlus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of the panel. GP1, MBP, and GP1-MBP are indicated.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance-5

Protein was eluted with 10 mM maltose, cleaved with Factor Xa, and purified by SEC. (A) Western blot of protein in (lane 2) amylose capture eluate, (lane 3) Factor Xa cleavage reaction, and (lanes 4–10) SEC fractions 4–10. The blot was probed with a rabbit α-MBP polyclonal antibody and then detected with an HRP-conjugated goat α-rabbit IgG antibody. (B) The Western blot in panel A was stripped, reprobed with LASV mAb mix containing NP-specific mAbs, and then detected with an HRP-conjugated goat α-mouse IgG antibody. The identity of each lane is the same as that indicated in Panel A. (C) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, (lane 5) SEC-purfied NP generated from pooled NP-containing fractions, and (lane 6) SEC-purified MBP. (Lane 1) SeeBluePlus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of each panel. NP, MBP, and NP-MBP are indicated.<p><b>Copyright information:</b></p><p>Taken from "Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance"</p><p>http://www.virologyj.com/content/5/1/74</p><p>Virology Journal 2008;5():74-74.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2435526.</p><p></p

Case fatality rates for suspected LF cases by ribavirin treatment status and serostatus.

<p>The presence of LASV Ag in serum of patients with observed survival outcomes and verified treatment status was assessed by recombinant Ag− and IgM-capture ELISA. Statistical significance for within and between group comparisons was determined using a multivariate logistic regression model (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s010" target="_blank">Table S9</a>). NS = not significant.</p

Age distribution of cases presenting to the KGH Lassa Ward, 2008–12.

<p>Panel A: Age distributions of patients presenting while antigenemic (Ag+/IgM±). Panel B: Age distributions of nonantigenemic patients presenting with serum anti-LASV IgM (Ag−/IgM+). Panel C: Age distributions of nonantigenemic patients presenting without anti-LASV IgM seropositivity (Ag−/IgM−). In Panels A–C yellow portion of bars represent patients who were discharged and black portion of bars represent patients who died. Panel D: Age demographic for the population of Sierra Leone (2010 estimate). Among patients who died, the age distributions differed significantly between the Ag+/IgM± and Ag−/IgM− groups (p = .005). Distributional comparisons were carried out using the Kolmogorov-Smirnov technique (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s006" target="_blank">Table S5</a>).</p

Suspected cases of LF evaluated at the KGH Lassa Laboratory and numbers of patients admitted to the KGH Lassa Ward, 2008–12.

<p>Non-admitted patients include those where only blood samples were submitted for screening from referral health-posts, patients dying en route to the hospital (DOA = dead on arrival), and patients not meeting the LF suspected case criteria (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd-0002748-t001" target="_blank">Table 1</a>). Characteristics of study patients are compiled in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s002" target="_blank">Table S1</a>.</p

Monthly distribution of suspected LF cases presenting to the KGH Lassa Ward by serostatus, 2008–2012.

<p>Panel A: antigenemic Lassa fever cases (Ag+/IgM±). Panel B: Patients with serum anti-LASV IgM (Ag−/IgM+). Panel C: Patients with no Lassa virus seropositivity (Ag−/IgM−). The monthly frequency distributions differed between each of the serostatus group comparisons as assessed using a Poisson regression model (p<.001 for all serostatus comparisons; data not shown).</p