Periplasmic production of Green Fluorescent Protein is poorly tolerated by Escherichia coli

Escherichia coli is a commonly used host for recombinant protein production. It is advantageous to direct many recombinant proteins, especially those that require disulphide bonding for function, such as antibody fragments, to the periplasm of E. coli. This requires N-terminal fusion of a signal peptide that directs the polypeptide chain through the relevant translocation apparatus. Signal peptides cannot be selected on the basis of recombinant gene sequence, so screening is required to select the optimal signal peptide for each product, typically using subcellular fractionation, a time-intensive process. Fusion of a fluorescent protein such as GFP to the C-terminal of recombinant proteins has previously been used to accelerate cytoplasmic protein production process development, but most GFP proteins are not active in the periplasm. Previous studies have developed GFP derivatives that fold rapidly (such as superfolder GFP, sfGFP) and have been reported to be periplasmically active. Here, we tested the applicability of sfGFP as a periplasmic screening tool using single-cell analysis and structured illumination microscopy. We discovered that sfGFP is very poorly tolerated in the periplasm, causing deleterious effects on E. coli physiology, manifesting as poor growth, cell death, and loss of recombinant protein productivity. A further reason for poor GFP functionality in the periplasm is errant disulphide bonding, so we tested a cysteine-free GFP, which cannot form disulphide bonds; results were similar to sfGFP. In conclusion, currently-available GFP variants are poor fusion partners for screening production and translocation of recombinant proteins to the E. coli periplasm due to their negative impact on physiology. HighlightsO_LIGFP is a useful screening tool recombinant protein production. C_LIO_LIWe tested periplasmic expression of GFP derivatives sfGFP and cfSGFP2. C_LIO_LIWe used structured illumination microscopy to visualise GFP accumulation. C_LIO_LIPeriplasmic GFP derivatives have significant negative effects on bacterial physiology C_LI