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Escherichia coli
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Hisashi Yasueda, Hiroshi Matsui
Many eukaryotic proteins can be produced in large amounts by E. coli in the form of hybrid fusion proteins that are constructed by fusing the foreign gene to the coding sequence of highly expressed genes original to E. coli itself or obtained from eukaryotes. With such constructs, the read-through of translation from the upstream gene ensures a high translational efficiency and resolves problems related to the initiation of translation. Coincidentally, the fusion products are frequently resistant to proteolytic degradation, thereby overcoming the instability problems that can be encountered with small peptides [1]. Furthermore, it is also possible to devise an additional amino acid extension sequence (tail) at the NH2-terminus or COOH-terminus to assist in purification. For instance, the use of fusion protein with β-galactosidase, protein A, maltose-binding protein (MBP), and glutathione-S-transferase (GST) allows affinity purification with TPEG, IgG, starch, and glutathione as the respective ligands [95-100].
Use of Recombinant DNA Technology for Engineering Mammalian Cells to Produce Proteins
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
The level of protein expression from heterologous genes introduced into mammalian cells depends on multiple factors including DNA copy number, efficiency of transcription, mRNA processing, mRNA transport, mRNA stability and translational efficiency, and protein processing, secretion, and stability. The rate-limiting step for high-level expression may be different for different genes. Controls at each one of these levels will be discussed in turn.
It's not just about protein turnover: the role of ribosomal biogenesis and satellite cells in the regulation of skeletal muscle hypertrophy
Published in European Journal of Sport Science, 2019
Matthew Stewart Brook, Daniel James Wilkinson, Ken Smith, Philip James Atherton
Protein synthesis is the process by which ribosomes create polypeptide chains through linking amino acids together in a specific order according to mRNA. As such, rates of protein synthesis can be modulated by the rate of mRNA translation, known as “translational efficiency”. A primary control point regulating translational efficiency and therefore protein synthesis in the majority of eukaryotic cells is by cap dependent translation. This involves the assembly of many eukaryotic initiation factors (eIF's) to form a preinitiation complex (PIC) that interacts with the 5′ end of an mRNA to instigate protein synthesis (for more detail readers are directed to [Jackson, Hellen, & Pestova, 2010]). However, with protein synthesis being an energy demanding processes (e.g. through peptide bonding) it is unsurprising that there is myriad of regulating signaling cascades, many of which culminate on the mammalian target of rapamycin (mTOR), that integrates signals such as exercise, AA availability and energy status to coordinate cellular metabolism (Goodman et al., 2011). Some of the best understood targets of mTOR are those directly involved in cap-dependent translation, including P70S6K1, 4E-BP1, and RPS6 that can enhance translation initiation and efficiency in the absence of ribosomal biogenesis (Chesley, MacDougall, Tarnopolsky, Atkinson, & Smith, 1992).
Preparation and self-cleavage of fusion soluble farnesyl diphosphate synthase in E. coli
Published in Preparative Biochemistry & Biotechnology, 2023
Wenfeng Ni, Zixuan Wang, Aifang Zheng, Ying Zhao
Escherichia coli is a widely used expression platform because of its well-studied genetic background, short generation cycle, and cost-effective culture methods.[17,18] In general, prokaryotic expression vectors contain the T7 promoter require bacteriophage T7 RNA polymerase for the transcription of target genes, which is more efficient than other RNA polymerases present in E. coli.[19,20] Several commercial E. coli strains harbor T7 RNA polymerase, such as BL21 (DE3) and Rosetta (DE3). BL21 (DE3) lacks the Lon and OmpT proteases, thereby substantially reducing recombinant protein degradation.[21] Rosetta (DE3) is an engineered strain that provides rare tRNAs for recombinant protein expression, which may increase translational efficiency and reduce metabolic delay.[22] Although the superabundant expression of recombinant proteins can achieve a high yield, Inclusion body (IB) formation is a problem that needs to be solved urgently.[23–28] However, multiple solutions are available, including the optimization of induction conditions, expression with molecular partners, and fusion with protein tags. Several fusion tags are typically chosen for the acquisition of soluble proteins, such as GB1-domain (GB1), the IgG-binding domain (ZZ), and disulfide bond A (DsbA), which facilitate the appropriate folding of target proteins.[29–32] Fusion tags can block catalytic sites and negatively influence the enzymatic activity of recombinant proteins. Some peptide fragments linked between the target proteins and fusion tags are recognized by different proteases to remove the superfluous tags, such as Tobacco Etch Virus protease (TEVp). Therefore, when combined with the cleavage of proteases, the activity and solubility of these proteins are satisfactory.