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

<p>Abstract</p> <p>Background</p> <p>There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2).</p> <p>Results</p> <p>Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of <it>Escherichia coli </it>(<it>E. coli</it>) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA).</p> <p>Conclusion</p> <p>These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.</p

Lassa Fever in Post-Conflict Sierra Leone

Background: Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the world's highest incidence of the disease. Methodology/Principal Findings Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leone's thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects. Conclusions/Significance: Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF

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-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-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-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-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-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