Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD
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BACKGROUND 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. RESULTS Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein MBP fusions in the Rosetta strains of Escherichia coli E. coli 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. CONCLUSION These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.
- Medicine and Medical Research