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Assay Requirements for Cell Culture Process Development
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
Mary B. Sliwkowski, Edward T. Cox
SDS gels can be used to monitor proteolysis, which is necessary for correct processing of many proteins, but can also be an unwelcome side effect of cell lysis during culture. Samples run with and without reducing agents, such as β-mercaptoethanol (βME) or dithiothreitol (DTT), also provide information on disulfide-bonded subunits and aggregates. Glycoproteins often appear as broad smears rather than sharp bands due to their more extensive heterogeneity.
Synthesis of PEGylated cationic curdlan derivatives with enhanced biocompatibility
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Muqier Muqier, Hai Xiao, Xiang Yu, Yifeng Li, Mingming Bao, Qingming Bao, Shuqin Han, Huricha Baigude
Dithiothreitol (DTT), also known as Cleland's reagent, is a small-molecule redox reagent that participates to disulfide exchange reactions [35]. In order to further confirm the successful conjugation of 2S PEG to 6AC-100, we used DTT to reduce the disulfide bond to release free amino group and enhance the binding ability of PEGylated 6AC-100 with siRNA. Briefly, different final concentration of DTT was added to stock solution (1 mg/mL) of PEGylated 6AC-100 and incubated at 37 °C for 2 h (DTT with final concentration of 500 mM was added to 1 mg/mL of 6AC-2S PEG5 and 6AC-2S PEG10, DTT with final concentration of 700 mM was added to 1 mg/mL of 6AC-2S PEG20 and 6AC-2S PEG40). Then, siRNA was added to the solution at various N/P ratio and the siRNA encapsulation efficiency of the polymers was analyzed using gel mobility shift assay as described previously.
Modeling and evaluation of the sucrose-degrading activity of recombinantly produced oligo-1,6-glucosidase from A. gonensis
Published in Preparative Biochemistry & Biotechnology, 2023
Hakan Karaoglu, Zeynep Dengız Balta
A series of enzyme reactions were performed at pH 7.0 at 60 °C to evaluate the effects of various metal ions and organic compounds on rAgoSuc2. The experiments without any metal ions or organic compounds were chosen as the control group. It was observed that Zn2+, Cu2+, Pb2+, Ag2+, Fe3+, Hg2+, and Al2+ metal ions inhibited the enzyme activity even at low concentrations (1 mM). Adding 1 mm Co2+ ion exhibited an activator effect on the enzyme, while adding higher concentrations of Co2+ (5 mM and 10 mM) showed an inhibitory effect on the enzyme. It was detected that Mn2+, Fe2+, and Mg2+ ions behaved as activators for the enzyme. Besides K+, Na+, Ba2+, and Ca2+ ions, they exhibited neither an activator nor inhibitory effect on the enzyme (Figure 9). While the use of 10 mM Sn2+ did not cause any inhibition on the enzyme, a weak inhibitory effect of Sn2+ was determined with the use of 1 mM and 5 mM of this metal ion. The results of this study are compatible with the literature. Similarly, Mn2+ and Mg2+ metal ions have been reported as activators on O-1-6-glucosidase of Thermo anaerobium Tok6-B1.[33] In addition to metal ions, many organic compounds were searched in terms of their activator and inhibitor effects on the enzyme (Figure 11). It was determined that ammonium sulfate, boric acid, urea, oxalic acid, sodium citrate, and EDTA did not have any inhibitory or activating effects on the enzyme. The fact that EDTA, a chelating agent, did not have any effect on the enzyme’s activity was evidence supporting the conclusion that the enzyme did not have a cofactor and existed as an apoenzyme. While Tween 80 and DTT showed a high activator effect, it was determined that methanol, ethanol, glycerol, acetone, SDS, and β-mercapto-ethanol inhibited the enzyme even at low concentrations (Figure 10). Kim et al. [34] reported that tween 80 exhibited an activator effect on carbohydrolase I (CBH I) of Trichoderma viride. It was interpreted that the activity of CBH I increased as tween 80 facilitated the interaction of the enzyme’s cellulose binding domain and the substrate.[34] Similarly, the addition of tween 80 could increase rAgoSuc2 activity by facilitating the interaction of rAgoSuc2’s carbohydrate binding domain with sucrose. Dithiothreitol (DTT) can reduce disulfide bonds in enzymes and mask the effects of the residue on the enzyme by reacting with cysteine residues.[35] According to the predicted tertiary structure of rAgosuc2 (Figure 2), it was clearly seen that there was a cystein residue (274th amino acid) in the active site of the enzyme. The increase in activity of rAgoSuc2 could be explained by the fact that DTT reacted with this cysteine residue and facilitated the binding of the substrate in the active site of the enzyme.