[PMID]: | 28569168 |
[Au] Autor: | Singh SK; Roeffen W; Mistarz UH; Chourasia BK; Yang F; Rand KD; Sauerwein RW; Theisen M |
[Ad] Endereço: | Department for Congenital Disorders, Statens Serum Institut, Artillerivej 5, 2300, Copenhagen, Denmark. |
[Ti] Título: | Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine. |
[So] Source: | Microb Cell Fact;16(1):97, 2017 May 31. |
[Is] ISSN: | 1475-2859 |
[Cp] País de publicação: | England |
[La] Idioma: | eng |
[Ab] Resumo: | BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. RESULTS: A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats. CONCLUSIONS: The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine. |
[Mh] Termos MeSH primário: |
Vacinas Bacterianas/biossíntese Vacinas Bacterianas/imunologia Imunogenicidade da Vacina/imunologia Lactococcus lactis/metabolismo Plasmodium falciparum/metabolismo Proteínas Recombinantes de Fusão/biossíntese
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[Mh] Termos MeSH secundário: |
Animais Vacinas Bacterianas/isolamento & purificação Reatores Biológicos Lactococcus lactis/genética Plasmodium falciparum/química Plasmodium falciparum/imunologia Subunidades Proteicas/biossíntese Subunidades Proteicas/química Subunidades Proteicas/isolamento & purificação Ratos Proteínas Recombinantes de Fusão/imunologia Proteínas Recombinantes de Fusão/isolamento & purificação
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[Pt] Tipo de publicação: | JOURNAL ARTICLE |
[Nm] Nome de substância:
| 0 (Bacterial Vaccines); 0 (Protein Subunits); 0 (Recombinant Fusion Proteins) |
[Em] Mês de entrada: | 1711 |
[Cu] Atualização por classe: | 171108 |
[Lr] Data última revisão:
| 171108 |
[Sb] Subgrupo de revista: | IM |
[Da] Data de entrada para processamento: | 170602 |
[St] Status: | MEDLINE |
[do] DOI: | 10.1186/s12934-017-0710-0 |
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