Utiliza este identificador para citar o vincular este elemento: http://hdl.handle.net/10553/35684
Títulos: Generation of nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins
Autores/as: Hu, Yaozhong
Romão, Ema
Vertommen, Didier
Vincke, Cécile
Morales-Yánez, Francisco
Gutiérrez Cabrera, Carlos 
Liu, Changxiao
Muyldermans, Serge
Clasificación UNESCO: 32 Ciencias médicas
Palabras clave: BL21 E.coli expression
IMAC
SlyD
Nanobody
Immunoprecipitation
SEC
Recombinant protein
Fecha de publicación: 2017
Revistas: Protein Expression and Purification 
Resumen: The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coll. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SIyD contaminant was quantitatively eliminated.
URI: http://hdl.handle.net/10553/35684
ISSN: 1046-5928
DOI: 10.1016/j.pep.2017.06.016
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