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Cell Biology for Bioprocessing
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Although some proteins are translocated into the ER after they are completely synthesized in the cytoplasm, most (including typical recombinant protein products) are translocated as nascent protein molecules (Figure 2.8, Panel 2.21). Proteins that are destined for secretion, the cytoplasmic membrane, and organelles have a leader sequence of five to thirty amino acids at the amino terminus. These signal sequences serve as markers of each protein’s destination. After the mature mRNA with the polyadenylated tail (polyA tail) is exported to the cytosol from the nucleus, a ribosome binds to the translation start site of the mRNA and starts translation. The signal peptide of the nascent protein is synthesized and then recognized by signal recognition particles (SRP) near the ER. The binding of SRP to the signal sequence causes a pause in translation and docks the nascent protein (which has only the beginning segment of the entire sequence) along with the ribosome and the mRNA to a translocator (also called a translocon or translocation channel) on the ER membrane. The nascent polypeptide is then transferred through the translocator into the ER lumen, but the ribosome and mRNA are left on the cytosolic surface of the ER. Subsequently, translation elongation resumes and the elongating polypeptide passes through the channel of the translocator into the ER lumen. As the translation proceeds downstream of the mRNA, new ribosomes continue to bind to the translation initiation site of the mRNA, initiating the synthesis of the signal peptide and repeating the process of translocating the nascent protein into the ER. Thus, on the cytoplasmic side of the ER each mRNA molecule has multiple ribosomes actively translating the protein next to the translocator.
Sustainability and Development of Industry 5.0
Published in Pau Loke Show, Kit Wayne Chew, Tau Chuan Ling, The Prospect of Industry 5.0 in Biomanufacturing, 2021
Hui Shi Saw, Abdul Azim bin Azmi, Kit Wayne Chew, Pau Loke Show
Short amino-terminal parts of exported precursor proteins, the signal peptides direct the export of respective proteins in the cytoplasmic membrane. Gram-positive bacteria, bacillus species that usually possess only a single cytoplasmic membrane can therefore export a target protein tagged with the corresponding signal across this major permeable barrier into the growth medium. In the effort of increased secretion of heterologous proteins by bacterial host organism, research on modification of existing signal peptides or de novo design of synthetic signal peptides is reported.
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
Only proteins that contain the signal peptide in their NH2-termini are allowed entry into the general secretory pathway in both prokaryotic and eukaryotic cells. The proteins are delivered to their proper sites according to the signal of the signal peptide. Whether the signal peptide can be cleaved or not, it is a determinant of the protein’s topology; locating in the membrane, anchoring in the membrane, or translocating across the membrane [1].
Enhanced periplasmic expression of human activin A in Escherichia coli using a modified signal peptide
Published in Preparative Biochemistry & Biotechnology, 2020
Zahra Hajihassan, Niloofar Khairkhah, Farshid Zandsalimi
Activins are a family of dimeric glycoproteins whose mature forms consist of homodimers (βAβA) or heterodimers (βAβB) of the β subunits.[1] Activin A is a homodimer type of activins linked by a single covalent disulfide bond (Cys80).[2] Activin A mature regions share nine conserved cysteines which substitution of either cysteine residues 44 and 80 will cause a monomer formation with 2% biological activity of wild type activin A.[2] Activin A plays many important roles in the body, including anti-inflammatory role,[3] wound repairing,[4] regulation of cell proliferation, apoptosis, carcinogenesis[5] and it’s a commitment factor in erythroid differentiation.[6] Because of its clinical applications, producing recombinant activin A is an excellent choice. Benefits of cost and easily use, make Escherichia coli one of the most widely used hosts to produce recombinant proteins. However, expression of recombinant proteins containing disulfide bonds such as activin A in E. coli often results in the formation of misfolded proteins or inclusion bodies.[7] Disulfide bond formation is impaired in the reducing environment of cytoplasm. Therefore, expression of proteins such as activin A in the oxidative periplasmic compartment is beneficial for correct formation of disulfide bonds. The periplasm of Gram-negative bacteria contains various folding modulators such as molecular chaperones and disulfide-isomerases (DSBs)[8] which facilitate correct disulfide bonds formation and protein folding.[9] Using appropriate signal peptide translocates the unfolded precursors into the periplasmic space of bacteria.[10] Although, there are many natural signal peptides which can lead the interest protein to the periplasmic space but none of them have 100% translocation efficiency, so minor modification of natural signal peptides may be beneficial for improved secretory expression. A typical signal peptide is made up of three functional domains including a positively charged N-region, a hydrophobic H-region, and a polar C-region.[11] Increasing the hydrophobicity levels in the H-core of the signal sequence for example by insertion of a hydrophobic residue like leucine may be an effective strategy to enhance the efficiency of secretory expression of interest proteins.[12] Herein, we increased the hydrophobicity level of the Iranian native Bacillus licheniformis α-amylase signal sequence to enhance the secretion of activin A into the periplasmic space and then, we cloned modified signal sequence and human activin A cDNA into the pET21a(+) expression vector.[13] Recombinant activin A produced and accumulated in the periplasmic space was then purified using immobilized metal affinity chromatography (IMAC) and its biological activity was tested on K562 cell line.