Protein production, purification, AVEXIS assays and SPR were performed essentially as described11 (link) except the type II proteins which were expressed with an N-terminal Cd4d3+4-biotin tag and a mouse antibody signal peptide. PfRh5 was expressed as above except that a non-endogenous signal peptide was added and the threonines in potential N-linked glycan sequons were mutated to alanine to prevent inappropriate glycosylation. All constructs were chemically synthesized and codon optimised for mammalian expression (Geneart AG). Purified pentameric proteins used in invasion assays were made by replacing the β-lactamase reporter in the prey plasmid with a hexa-his tag, purified and buffer exchanged into RPMI prior to use. BSG variants were produced by site directed mutagenesis.
Protocol detail
Protein Expression and Purification for Invasion Assays
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Alanine
Antibody
Assays
Biotin
Buffer
Codon
Glycan
Glycosylation
Hexa
Mammalian
Mouse
Plasmid
Proteins
Signal peptide
Site directed mutagenesis
Threonines
β lactamase
Corresponding Organization :
Other organizations : Wellcome Sanger Institute, Harvard University, Japanese Red Cross Society, Japan, Cheikh Anta Diop University
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Variable analysis
independent variables
- Type of proteins expressed (type II proteins with N-terminal Cd4d3+4-biotin tag and mouse antibody signal peptide, PfRh5 with non-endogenous signal peptide and threonine to alanine mutations in potential N-linked glycan sequons)
- BSG variants produced by site directed mutagenesis
- Protein production
- Protein purification
- AVEXIS assays
- Protein production, purification, AVEXIS assays and SPR protocols followed as described in the reference (except for the modifications to the type II proteins and PfRh5).
- All constructs were chemically synthesized and codon optimised for mammalian expression.
- Purified pentameric proteins used in invasion assays were made by replacing the β-lactamase reporter in the prey plasmid with a hexa-his tag, purified and buffer exchanged into RPMI prior to use.
- None specified.
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