One problem that arises with reporter proteins is that they only function in specific places of the cell. For example, alkaline phosphate and B-lactamase are two reporter proteins that can only function in the periplasmic space. This means that they can only identify locations of proteins in the periplasmic space. However, B-Galactosidase, another reporter protein, can only identify proteins when they are in the cytoplasm. Through the use of mutations caused by inactive B-Galactosidase in regions of the cell other than the cytoplasm, sec genes can be identified and located which are responsible for encoding information about export machinery.
However, B-Galactosidase can sometimes cause protein export issues. When hybrid proteins over-synthesize B-Galactosidase, it causes a back up at the export pathway because the high amount of B-Galactosidase is incompatible with the export machinery. This is called overproduction lethality. This is a major issue because the export pathway becomes severely jammed and normal proteins are unable to be exported.
To better understand bacterial protein export, there would be a reporter protein that is active in the cytoplasm, and can travel freely across the export pathway so there is no jamming. GFP was used to investigate this new type of reporter protein. GFP is a good solution because the active form of the...
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...t proteins were still able to pass through. However, since the hybrid protein localized in the periplasmic space did not glow, a mutant form of the malE-gfp gene could be used to isolate export mutants. Mutant forms of malE in pMGP2 were cut with restriction enzyme 2-kb EcoRV-BgII and put back into a non-mutant form of pMGP2. All of the recombinants glowed, meaning that GFP can be used as a tool to isolate and find new export mutants.
In conclusion, it was found that the MBP-GFP hybrid protein glowed and was active in the cytoplasm, but did not glow and was not active when exported out of the cytoplasm. Unlike B-Galactosidase, an overproduction of the hybrid protein did not cause jamming. This is beneficial because in the future, instead of using B-Galactosidase as a reporter protein in the cytoplasm, GFP can be used as a tool to isolate and study export proteins.
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