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Introduction and Background
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
An example of a prokaryotic cell is molecular biology’s key organism, the bacterium Escherichia coli, usually just called E. coli. The structure of an E. coli cell is shown in Figure 1.11a,b; it consists principally of a cell wall, cell membrane, circular DNA chromosome packaged into a nucleoid region, and ribosomes. Within its fluid contents or cytoplasm are all of the enzymes needed to replicate DNA and transcribe DNA to RNA. RNA is translated to protein in the ribosomes. The cytoplasm also contains disequilibrium concentrations of ions, particularly potassium, leading to a nonzero membrane potential (see Chapter 15 for more on the origin and measurement of membrane potentials). The space between the membrane and cell wall is called the periplasm and may comprise 40% of the cell’s volume; many important reactions, such as neutralization of antibiotics, occur in this space. The structure of the E. coli membrane and periplasm is characteristic of the class of bacteria called Gram negative (Figure 1.11c).
Transport of Nutrients and Carbon Catabolite Repression for the Selective Carbon Sources
Published in Kazuyuki Shimizu, Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production, 2017
The gram-negative bacteria such as E. coli have two concentric membranes surrounding the cytoplasm, where the space between these two membranes is called as periplasm. The outer membrane and inner or cytoplasmic membrane constitute a hydrophobic barrier against polar compounds. The outer membrane contains channel proteins, where the specific molecules can only move across these channels. In the outer membrane of E. coli, 108 channels are formed by the porin proteins (Nikaido and Nakae 1980). Porinsare the outer membrane proteins that produce large, open but regulated water-filled pores that form substrate-specific, ion-selective, or nonspecific channels that allow the influx of small hydrophilic nutrient molecules and the efflux of waste products (de la Cruz et al. 2007). They also exclude many antibiotics and inhibitors that are large and lipophilic (Nikaido 2003). Porins including OmpC and OmpF of E. coli form stable trimers with a slight preference for cations over anions (Saier et al. 2006, Saier et al. 2009). The OmpC and OmpF are the most abundant porins present under typical growth condition representing up to 2% of the total cellular protein (Nikaido 1996). OmpF seems to have slightly larger channel than OmpC, where these are the constitutive porins. Their relative abundance changes depending on such factors as osmolarity, temperature, and growth phase (Hall and Silhavy 1981, Lugtenberg et al. 1976, Pratt and Silhavy 1996). These porins serve for glucose to enter into the periplasm when glucose is present at higher concentration than about 0.2 mM (Nikaido and Vaara 1985, Death and Ferenci 1994). The diffusion rate of glucose molecule is about two-fold higher through OmpF than through OmpC (Nikaido and Rosenberg 1983). Under glucose limitation, the outer membrane glycoporin LamB is induced (Death and Ferenci 1994), where this protein permeates several carbohydrates such as maltose, maltodextrins, and glucose (von Meyenburg and Nikaido 1977), where about 70% of the total glucose import is contributed by LamB (Death and Ferenci 1994). Glucose transport by diffusion through porins of the outer membrane is a passive process (Gosset 2005).
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
The current efforts to capitalize bacterial IBs without compromising product yield principally encompasses strategies to translocate the newly formed IBs from the reducing cytoplasmic environment to relatively more oxidative periplasmic or extracellular space.[80] The underlying reason is several advantages the periplasmic and extracellular space offers during the expression of functional therapeutic proteins. For instance, periplasmic space confers resistance to the protease-mediated degradation of the target proteins.[47] Further, it streamlines downstream processing because very few naturally occurring cellular proteins are present in E. coli periplasm[81] (Figure 3).
Lindane degradation by root epiphytic bacterium Achromobacter sp. strain A3 from Acorus calamus and characterization of associated proteins
Published in International Journal of Phytoremediation, 2019
Polypeptide band 1 of Achromobacter sp. strain A3 was identified as alpha/beta hydrolase fold-3 domain-containing protein. This protein has hydrolase activity and takes part in metabolic processes. Hydrolases catalyze the hydrolysis of various bonds. Here, in our study, the expression of this protein solely under the stress of lindane proves that it is involved in lindane hydrolysis. Other studies show that LinB from Sphingomonas paucimobilis UT26 is a haloalkane dehalogenase belonging to α/β-hydrolase family. These are microbial enzymes which catalyze the hydrolytic dechlorination reaction in the degradation of lindane. These enzymes play important role in bioremediation of contaminated areas, hence, can be used for the protection of the environment (Nagata et al. 1997; Kmunicek et al. 2005). Polypeptide band 2 was identified as extracellular solute-binding family protein. The molecular function of this protein is transporter activity, i.e., it enables the aimed movement of substances such as ions, macromolecules, and small molecules into or out of the cell. In gram-negative bacteria, solute binding proteins are dissolved in the periplasm and participate in transmembrane transport. In the present study, the expression of this protein solely under the stress of lindane demonstrates its role in transmembrane transport of lindane for utilization of lindane by bacteria. In Sphingomonas paucimobilis UT26, genes for ABC-transporter system are essential for utilization of lindane (Endo et al. 2007). It has also been found that periplasmic phosphate binding protein initiates the signal resulting in induction of phosphate regulon in enteric bacteria (Tam and Saier 1993).
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.