|Ex λ||Em λ||EC (M-1 cm-1)||QY||Brightness||pKa||Maturation (min)||Lifetime (ns)|
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We found that [adding F99S and V163A to EBFP2] did not prevent oligomer formation, but we observed a significant reduction in the proportion of covalent oligomers compared with monomeric species... Therefore, it appeared that we would need to mutate the two FP cysteine residues (C48 and 70). However, mutagenesis of the cysteines in the closely related EGFP decreased brightness with single cysteine mutations and rendered the protein dark upon mutating both cysteines... In an attempt to restore fluorescence, we turned our attention to the missing F99S and V163A cycle-3 mutations. Additional rounds of mutagenesis revealed that the combination of C48S, C70S and V163A resulted in a BFP with brightness comparable to parental EBFP2, without the problematic cysteine residues
The new BFP, termed oxBFP, is fluorescent within the cytoplasm and lumen of the ER and does not participate in non-native disulphide bonds