Smic proteins [24-28]. The latter two proteins also display X-Pro isomerization
Smic proteins [24-28]. The latter two proteins also display X-Pro isomerization activity but their function in assisting EntinostatMedChemExpress Entinostat protein folding has been attributed primarily to their role as chaperones [29,30]. DnaK/DnaJ/GrpE, GroEL/GroES and ClpB can function synergistically in assisting protein folding and therefore expression of these chaperones in combinations has been shown to be beneficial for protein expression [11,31].1.3 Expression of disulfide-bonded proteinsTargeted strain engineering focuses on the introduction of mutations in DNA sequences known to affect protein synthesis, degradation, secretion or folding. SeveralMany biotechnologically important proteins contain disulfide bonds. The cytoplasm of E. coli is normally maintained in a reduced state that precludes the formation of disulfide bonds via the action of the thioredoxin and glutaredoxin/glutathione enzyme systems [32]. Therefore, proteins with disulfides normally need to be exported into the periplasm. In the periplasm, disulfide bond formation and isomerization is catalyzed by theMakino et al. Microbial Cell Factories 2011, 10:32 http://www.microbialcellfactories.com/content/10/1/Page 3 ofDsb system, which comprises DsbABCD and G. Coexpression of the cysteine oxidase DsbA, the disulfide isomerase DsbC or combinations of the Dsb proteins, have been employed for the successful expression of numerous heterologous proteins such as scFvs, plasminogen activators, human nerve growth factors and others [25,33-35]. Mutant strains defective in glutathione reductase (gor) or glutathione synthetase (gshA) together with thioredoxin reductase (trxB) render the cytoplasm oxidizing. These strains are unable to reduce ribonucleotides and therefore cannot grow in the absence of exogenous reductant, such as dithiothreitol (DTT). Suppressor mutations in the gene ahpC, which encodes the peroxiredoxin AhpC, allow the channeling of electrons onto the enzyme ribonulceotide reductase enabling the cells to grow in the absence of DTT. In such strains, exposed protein cysteines become readily oxidized in a process that is catalyzed by thioredoxins, in a reversal of their physiological function, resulting in the formation of disulfide bonds. A number of heterologous multi-disulfidebonded proteins have been produced in the cytoplasm of E. coli FA113 cells (trxB gor ahpC*) or OrigamiTM (Novagen) at high yields [36]. Additionally, it was recently shown that bacterial strains with different mutations in the thioredoxin/thioredoxin reductase and glutaredoxin/glutathione reductase genes and containing different suppressor mutations in alleles of ahpC, display dramatic differences in the kinetics of cysteine oxidation in the cytoplasm and in the yield of correctly folded proteins [28,37]. Very recently, Ruddock and colleagues have shown that overexpression of the sulfhydryl oxidase Erv1p from the inner membrane space of yeast mitochondria enables high-level production of a variety of complex, disulfide-bonded proteins of eukaryotic origin in the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28461567 title=View Abstract(s)”>PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28461585 cytoplasm of E. coli [38]. Remarkably, these investigators found that disulfide bond formation upon Erv1p coexpression could take place even in the absence of trxB gor mutations [39].1.4 Glycoprotein production in E. colifor the incorporation of diverse glycan structures onto a target polypeptide. Furthermore, forward engineering using shotgun proteomics and metabolic flux analysis has been applied to significantly improve the efficiency of protein glycosylation in.
