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Chemical Permeabilization of Cells for Intracellular Product Release
Published in Juan A. Asenjo, Separation Processes in Biotechnology, 2020
Thomas J. Naglak, David J. Hettwer, Henry Y. Wang
Inclusion bodies are easily recovered from inside host cells by lysis followed by low-speed centrifugation to remove cell debris. Washing and high-speed centrifugation can be used to purify the inclusion bodies as they are easily pelleted, although the inclusion body itself can contain more than one protein (Webster and Rementer, 1980). Inclusion body proteins are brought into aqueous solution using chaotropic agents such as 6 M guanidine hydrochloride or 8 M urea or anionic detergents such as sodium dodecyl sulfate. The chaotropes act to alter the structure of water, weakening the hydrophobic forces holding the proteins in aggregates. Detergents solubilize proteins by binding to them, the hydrophobic portions of the detergent molecules masking the hydrophobic portions of the protein molecules. This process is usually carried out in the presence of reducing agents such as β-mercaptoethanol. Under these conditions, proteins are completely denatured and dissociated into subunits, and all disulfide bonds are broken. Once in solution, the proteins can be purified by most standard techniques, with a few notable exceptions. For example, ionic detergents can interfere with some types of chromatography. When carrying out these procedures, it is often necessary to include the appropriate denaturant to prevent reaggregation, especially in the case of amphipathic proteins. To avoid reaggregation, a strong chaotrope such as guanidine or thiocyanate can sometimes be replaced by a weaker chaotrope such as urea (Moldow et al., 1972).
Medium Design for Cell Culture Processing
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
In addition to physiological antioxidants like vitamins C and E and glutathione, non-physiological antioxidant compounds are often included in culture media to minimize oxidation of labile medium components in culture fluid. β-mercaptoethanol is routinely used in the cultivation of stem cells and differentiated cells. Caution should be taken when using reducing agents in a medium if secreted proteins containing disulfide bonds are being produced. Mercaptoethanol can reduce the disulfide bridge to free thiols, which may then become reoxidized and form incorrectly paired disulfide bonds.
Electrophoresis
Published in Thomas J. Bruno, Paris D.N. Svoronos, CRC Handbook of Basic Tables for Chemical Analysis, 2020
Thomas J. Bruno, Paris D.N. Svoronos
Phenol, chloroform 2-mercaptoethanol, and also sodium dodecylsulfide (SDS) are also commonly used in electrophoresis. Phenol is corrosive and toxic; chloroform is toxic and a suspected carcinogen. Information on SDS, an anionic detergent applied to protein gels, is covered in the surfactants section in Chapter 14 of this book. 2-Mercaptoethanol (2-sulfanylethan-1-ol) is toxic, causing irritation to the nasal passageways and respiratory tract.
Biochemical characterization of a partially purified protease from Aspergillus terreus 7461 and its application as an environmentally friendly dehairing agent for leather industry
Published in Preparative Biochemistry & Biotechnology, 2021
Emmly Ernesto de Lima, Daniel Guerra Franco, Rodrigo Mattos Silva Galeano, Nelciele Cavalieri de Alencar Guimarães, Douglas Chodi Masui, Giovana Cristina Giannesi, Fabiana Fonseca Zanoelo
The reducing agents β-mercaptoethanol and DTT had no influence on protease activity. β-mercaptoethanol is known to promote the dissociation of protein disulfide bonds responsible for stabilizing the protein's tertiary structure, and consequently, it is known as a denaturing agent. The concentration of β-mercaptoethanol used in this work was not sufficient to promote the reduction of protease activity, suggesting that the enzyme has a high tolerance to the denaturing effects of β-mercaptoethanol and DTT, showing potential to be used in biotechnological processes that employ reducing agents in the process, such as in the treatment of bovine leather.[52] The protease of A. clavatus ES1[34] was also resistant to the presence of β-mercaptoethanol. DTT and β-mercaptoethanol did not affect the protease activity significantly from the fungus Myceliophthora sp.[53] Studies with A. terreus protease, on the other hand, had reduced activity in the presence of DTT at a concentration of 20 mM.[33]
Purification, biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001
Published in Preparative Biochemistry and Biotechnology, 2018
Manoj Kumar Narasimhan, Selvarajan Ethiraj, Tamilarasan Krishnamurthi, Mathur Rajesh
The effect of inhibitors was studied with 5 mM EDTA, 5 mM 2-mercaptoethanol, and 5 mM PMSF. EDTA chelates metal ions inhibiting metalloproteases. 2-Mercaptoethanol reduces disulfide bonds to disrupt the activity of enzymes. However, it may also reduce the active site thiol residues enhancing cysteine proteases activity. PMSF irreversibly sulfonates the active site serine residue to inhibit serine protease activity. The results show that residual FEA was found to be unaffected in the presence of EDTA and 2-mercaptoethanol, suggesting that isolated enzyme is neither metalloprotease nor cysteine protease, respectively, and this also indicates that disulfide bonds are not necessary for enzyme activity (Table 2). The results also show that incubation with PMSF leads to inhibition of FEA, suggesting that the isolated enzyme is a serine protease. It is interesting to note that several investigators have also reported that most fibrinolytic enzymes are serine proteases.[525354]