Applications of catalyzed cytoplasmic disulfide bond formation

Abstract

Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories

“…CyDisCo, either as a single polycistronic plasmid-based system or more commonly as a dual plasmid-based system, has been used to successfully produce a range of eukaryotic proteins having typically between one and five disulfide bonds (reviewed in [19]). The successful production of these correctly folded proteins with the help of co-or pre-expression of Erv1p and PDI in the cytoplasm of E. coli, made us consider what the limitations might be in terms of the production of more complex disulfide bonded proteins.…”

“…The mucus layer is also an interesting study subject from the pharmacological point of view, as drugs need to interact with and penetrate the layer in order to reach their targets [24]. Alpha tectorin on the other hand is present in the tectorial membrane of the ear canal and plays a significant role in the propagation of sound [19]. Even though these proteins have different functions both of them contain four VWF-D domains.…”

“…The alpha tectorin (P707-P981) construct, with 283 amino acids (8 disulfide bonds) lies at the top end of proteins previously reported to be produced using CyDisCo [19]. In contrast, the cysteine-rich mucin 2 (V36-G389) construct which contains 15 disulfide bonds in the native state, both contains more disulfide bonds than previously reported to have been made in any protein using CyDisCo and contains a higher disulfide density (8.5% of amino acids are Cys found in disulfide bonds).…”

“…A wide variety of prokaryotic and eukaryotic proteins containing disulfide bonds have been successfully produced using this system. However, most proteins reported to date as being produced have between one and five disulfide bonds (reviewed in [19]). The most complex protein reported to date was resistin, a homodimer with five disulfides in each monomer plus an inter-molecular disulfide [19].…”

“…However, most proteins reported to date as being produced have between one and five disulfide bonds (reviewed in [19]). The most complex protein reported to date was resistin, a homodimer with five disulfides in each monomer plus an inter-molecular disulfide [19]. This level of disulfide complexity is far below the level which can be produced in eukaryotic systems, with some extracellular mammalian proteins having in excess of one hundred disulfide bonds.…”