Recombinant PNGaseF production and deglycosylation assays

Recombinant PNGaseF production and deglycosylation assays. Open Lab notebook. 1.5 L of BL21 E.coli cells harbouring the pOPH6:EmPNGaseF vector encoding a His-tagged construct of Elizabethkingia miricola PNGase F (EmPNGaseF, Uniprot P21163, PNGF_ELIMR) were used to express the protein. EmPNGAse F was purified from the E.coli lysate by immobilised metal affinity and size exclusion chromatography and its activity assayed against 5 distinct glycoproteins.

The pOPH6:EmPNGaseF vector was a kind gift from Dominic Alonzi, Department of Biochemistry, Oxford University and Sarah Need, Open University. Glycoproteins from Johan Hill, Hubert Brandstätter, Marco Trerotola. PR is funded by a LISCB - UK Wellcome Trust ISSF Grant 204801/Z/16/Z. AL is funded by a PhD Studentship by the Italian Government.

Related Organizations

University of Leicester
United Kingdom

Keywords

PNGase F, pOPH6, Elizabethkingia miricola, P21163, IMAC, Size exclusion chromatography

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