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Cell Biology for Bioprocessing
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Folding of the polypeptide starts immediately upon translocation into the ER lumen. The signal peptide on the elongating polypeptide in the ER lumen is cleaved upon entry into the ER. A class of ER chaperones and other proteins that facilitate protein folding act on the nascent protein molecules to prevent them from aggregating and to facilitate folding. Their actions require cellular energy (ATP). An important member of the ER luminal chaperones, BiP (also known as GRP78), is also a component of the translocator complex. BiP binds to hydrophobic amino acids in incompletely folded proteins to prevent them from aggregating. In addition to BiP, major ER luminal chaperones include protein disulfide isomerase (PDI), which facilitates the formation of the correct disulfide bond.
Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli
Published in Preparative Biochemistry & Biotechnology, 2020
Inamur Rahman, Lina Fang, Zhang Wei, Xiaodong Zheng, Lian Jiazhang, Lei Huang, Zhinan Xu
Over the last decade, many scientists have devised an ideal and effective fusion expression system for soluble expression and purification of the target protein. These major fusion tags include MBP,[25,26] GST,[8,9] disulfide bond oxidoreductase (DsbA),[27] N-utilization substance protein A (NusA),[26] Trx,[1,17] protein disulfide isomerase (PDI), His6,[28] SUMO,[10] and b′a′domain of human PDI (hPDIb′a′).[23] Fusion expression strategies have resulted in improved hbFGF expression, but challenges still remained, such as insufficient fusion tag cleavage and low final product yield. In general, an ideal fusion expression tag should be highly efficient in improving soluble expression and folding, facilitate purification, be resistant to protease degradation, be easy to remove, have no negative effect on the functional bioactivity of targeted protein, and be generally applicable for a large number of proteins.
Antioxidant response mechanism of freshwater microalgae species to reactive oxygen species production: a mini review
Published in Chemistry and Ecology, 2020
Adamu Yunusa Ugya, Tijjani Sabiu Imam, Anfeng Li, Jincai Ma, Xiuyi Hua
The endoplasmatic reticulum is an organelle present in freshwater microalgae cell, which is responsible for the synthesis of protein and lipid [32,33]. It consists of a network of tubules that enable it to perform many versatile functions in the cell including protein folding, biosynthesis, translocation and post-translational modification such as glycosylation, disulfide bond formation, etc [34]. The ROS are produced in the endoplasmatic reticulum of freshwater microalgae during disulfide bond formation between polypeptide chain substrate [34]. The process of ROS formation in the endoplasmatic reticulum of freshwater microalgae involves a protein called protein disulfide isomerase (PDI) which is an enzyme found in the endoplasmatic reticulum of freshwater microalgae [35]. This enzyme catalyses the formation and breakage of disulfide bonds that is present between cysteine residues of proteins during folding. Hydrogen peroxide is produced in endoplasmatic reticulum when two electrons are transferred to the cysteine residual leading to the reduction of PDI active site while the polypeptide substrate is oxidised leading to the reduction of molecular oxygen into oxidant H2O2 [36]. The reduction of PDI active site is as a result of the interaction of PDI and ER Oxidoreductin 1 (Ero1) [37]. The Ero 1 is an oxidoreductase enzyme that catalyzes the formation and isomerisation of disulfide bond in endoplasmatic reticulum [35].
Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis
Published in Preparative Biochemistry and Biotechnology, 2019
Hyung-Min Lee, Se-Woong Park, Sung-Jun Lee, Kwang-Hoon Kong
We recently reported the production of recombinant brazzein in the yeast Kluyveromyces lactis, which has many advantages such as the fact that it is easy to scale up and is generally recognized as safe (GRAS).[13] The secretion of recombinant brazzein containing four disulfide bonds by K. lactis was enhanced by increasing the activities of protein disulfide isomerase and endoplasmic reticulum membrane-associated protein Ero1p.[14] This expression system provides a valuable tool for the commercialization of brazzein—for instance by the food industry[13,15,16]—as long as the production cost can be reduced by improving expression levels and purification yield.