Expression of biological active disulfide rich proteins using a wheat germ cell-free extract

Although disulfide bond formation in proteins is one of the most common types of post-translational modifications, the production of recombinant disulfide rich proteins remains a challenge. Most extracellular proteins are stabilized by multiple disulfide bonds formed by the oxidation of pairs of cysteine residues. Protein folding and disulfide bond formation are coupled, also known as oxidative protein folding. The most popular host for recombinant protein production is Escherichia coli, but disulfide rich proteins are here often misfolded, degraded, or found in inclusion bodies. As a consequence, the alternative cell-free protein synthesis based on wheat germ extract has become one of the standard protein production methods. This open system of cell-free expression using wheat germ embryos is ideal for the expression of disulfide rich proteins especially those from eukaryotes. This cell-free expression system is eukaryotic from origin, and as such, expected to synthesize multidomain proteins in a folded state. Also, it has the additional advantage that it is an open system in which the optimized mix of thiol-disulfide oxidoreductases or chaperones can be added, so that disulfide-rich proteins have no chance to aggregate the moment the polypeptide chain emerges from the ribosomes. In my thesis, I have optimized an in vitro wheat germ translation system for the expression of three important eukaryotic proteins that have to form multiple disulfide bonds. In chapter 1, I introduce you to all the available cell-free expression systems in full details with a special focus on the wheat germ and E. coli extracts. The mechanism of disulfide bond formation during oxidative protein folding as well as the oxidoreductases and the chaperones involved are being discussed. Further, I present the disulfide rich proteins that I have been studying and the objectives of this study. In chapter 2, I show the soluble expression of Mouse Found in Inflammatory Zone (mFIZZ1). mFIZZ1 expression in wheat germ extract results in soluble, but biological inactive protein. However, expression in combination with human quiescin sulfhydryl oxidase (hQSOX1b), the disulfide bond-forming enzyme of the endoplasmic reticulum, results in soluble, intramolecular disulfide bonded, monomeric, and biological active protein. The recombinant mFIZZ1 protein clearly suppresses the production of the cytokines IL-5 and IL-13 in mouse splenocytes cultured under Th2 permissive conditions. The quiescin sulfhydryl oxidase hQSOX1b seems to function as a chaperone and oxidase during the oxidative folding. In chapter 3, I optimized an in vitro wheat germ translation system for the expression of the highly toxic Androctonus australis hector (AahII) scorpion toxin, which has to form four disulfide bonds. I showed for the first time that recombinant N-terminal GST-tagged rAahII toxin is solubly expressed in an in vitro translation system. Although four disulfide bonds between consecutive and non-consecutive cysteines need be correctly formed, co-translation with oxidoreductases (hQSOX1b or hPDI) did not increase the final yield. After proteolytical cleavage of the GST-tag, recombinant rAahII toxin showed a high toxicity with an LD50-value of 30 ng for mice after intracerebroventricular (icv) injection. Further, the nanobody NbAahII10 raised against the native AahII toxin recognizes a structural epitope on recombinant toxin, which indicates that rAahII expressed with a wheat germ in vitro translation system is correctly folded. Moreover, NbAahII10 neutralizes the toxicity of recombinant rAahII for all injected mice. I showed for the first time the recombinant ex