Expression of BtrFicTA for AMPylation assays
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چکیده
We previously expressed VbhTA constructs for lysate AMPylation assays from the pRSFDuet-1 backbone, a plasmid with two multiple cloning sites that each enable the expression of constructs from a PT7 promoter. Our constructs encoded either the VbhT toxin alone (with an N-terminal hexahistidine (His6-)tag) to be expressed from the first multiple cloning site or had additionally the VbhA antitoxin (with an N-terminal hemagglutinin (HA-)tag) cloned into the second multiple cloning site [1]. However, we continuously failed to express soluble BtrFicT using the same procedure. Accidentally, we constructed a pRSFDuet-1 derivative encoding His6-btrficT in the first multiple cloning site and HA-btrficA in the second multiple cloning site in a way that an additional ATG start codon had been inadvertently placed between the ribosome binding site (RBS) and the ATG start codon of the HA-tag of HA-btrficA, but not in frame (plasmid pAH134b; see illustration below).
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