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Fondamental Aspects of Secretory Enzyme Production by Recombinant Microbes
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Noboru Takizawa, Mitsuo Yamashita, Yoshikatsu Murooka
The signal peptidase and the signal peptide peptidase are also indispensable for protein translocation. A periplasmic, an outer membrane, and a secretory protein are removed from the signal peptide by the signal peptidase, released from the plasma membrane, and exported to other pathways. Two signal peptidases have been found in E. coli, and each enzyme is located in the inner membrane. The catalytic site of the enzyme is exposed to the periplasmic surface [36-39]. The signal peptidase I has a wide spectrum of activity and digests between alanine and any amino acid residues, except a proline [40,41], whereas the signal peptidase II is specific for lipoproteins and digests between alanine or glycine and the glyceride-fatty acid-modified cysteine residues [42].
Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate
Published in Preparative Biochemistry & Biotechnology, 2023
Amuliya Kashyap, Kuldeep Saini, Meenu Saini, Yogender Pal Khasa, Rani Gupta
The lipase in the Lip+ host was expressed under the constitutive GAP promoter as an extracellular secretory protein by virtue of an α-secretory signal peptide at its N-terminus.[23] Therefore, lipase present in the extracellular culture broth of Lip+ host would result in hydrolysis of methyloleate into methanol and oleic acid, later of which is utilized by the host as a carbon source. However, sustained release of methanol could inhibit oleic acid utilization due to its catabolite repression[24] in the presence of methanol. It was crucial to confirm oleic acid utilization by the P. pastoris host in presence of methanol before examining the expression system. Therefore, wild-type X33 host was subjected to mixed-fed cultivation to study catabolite repression of oleic acid utilization in the presence of methanol. A methanol concentration of 1% (supplemented every 12 h) was selected because methanol released from methyloleate hydrolysis would never reach this high concentration, and moreover, higher concentrations of methanol are toxic to cellular viability.[2] Additionally, in a previous study, high methanol concentration (2%) was shown to be repressive for oleic acid utilization.[14]
Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line
Published in Preparative Biochemistry and Biotechnology, 2019
Mehri Mortazavi, Mohammad Ali Shokrgozar, Soroush Sardari, Kayhan Azadmanesh, Reza Mahdian, Hooman Kaghazian, Seyed Nezamedin Hosseini, Mohammad Hossein Hedayati
Endoplasmic reticulum is the prominent site of membrane and secretory protein folding. Protein folding is caused by molecular chaperones in ER such as glucose regulated protein 78/immunoglobulin binding protein (GRP78/BiP), X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), and activating transcription factor 6 (ATF6).[1] Throughout protein synthesis by ribosomal residues on ER, a quality control process and post-translational modifications take place.[2–4] Recombinant protein overexpression in transfected cell lines usually results in protein misfolding, ER stress, and protein aggregation,[5,6] leading to the unfolded protein response.[7] Molecular chaperones and protein folding enzymes in ER act to solve this problem and help the protein to find its folding.[8] ER stress is induced due to the overload of unfolded, misfolded, and mutate proteins that cannot pass the protein secretion pathway and are stopped in ER.[4] Unfolded protein response (UPR) occurs in case ER stress is sensed by molecular chaperones in ER such as IRE1, PERK, and ATF6. Then GRP is released to reduce protein misfolding, and, at that time, UPR is activated. XBP1 and ATF4 are two other molecular chaperones in ER which are involved in UPR pathway.[1,7]