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Brief introduction in Fluorescent Proteins

Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.

Overview of sfGFP

A mutant of the wild-type green fluorescent protein from Aequorea victoria, called super folder GFP (sfGFP), is a new and robust derivation, designed for in vivo high performance analysis of protein expression levels. It demonstrates increased stability at higher temperatures and is able to tolerate protein tagging to poorly folding proteins while still maintaining fluorescence. It contains the important S65T mutation and F64L for red shift and folding respectively while it also has six additional mutations for enhanced folding: S30R, Y39N, N105T, Y145F, I171V and A206V. The absorption peak is at 480 nm while its emission peak is around 510 nm. (Cotlet et. al 2006)

sfGFP expression in Bacillus Subtillus

sfGFP was codon optimized for Streptococcus pneumoniae and cloned into Bacillus subtillus. For sfGFP we used the Streptococcus pneumoniae codon adapted version, which was previously described to work best for B. subtilis (Overkamp et al. 2013).

Signal Peptide

References:

Overkamp, W. et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79, 6481–6490 (2013).



Sequence and Features