Optimization of cold-adapted alpha-galactosidase expression in Escherichia coli

A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296

“…It was also found that the alkaline phosphatase activity of the recombinant CamPhoD increased when the cells were cultured in a medium depleted by phosphorus. The growth of the recombinant E. coli cells in a MX medium containing 80 mM KH2PO4 described earlier [46] significantly reduced the alkaline phosphatase activity of CamPhoD down to 0.035 U/mg compared The values of CamPhoD-specific activities corresponded to the activities of the PhoD phosphatase/phosphodiesterase from Aphanothece halophytica and alkaline phosphatase from Vibrio sp., whose molecular weights were also similar to CamPhoD and other alkaline phosphatases [10,[41][42][43][44][45]. Thus, the molecular weights of the alkaline phosphatases' monomers from Streptomyces griseus IMRU 3570, Pyrococcus abyssi, and Thermotoga maritima were 62 kDa, 54 kDa, and 45 kDa, respectively [42][43][44].…”

“…It was also found that the alkaline phosphatase activity of the recombinant CamPhoD increased when the cells were cultured in a medium depleted by phosphorus. The growth of the recombinant E. coli cells in a MX medium containing 80 mM KH 2 PO 4 described earlier [46] significantly reduced the alkaline phosphatase activity of CamPhoD down to 0.035 U/mg compared with the activity of the enzyme, which was produced in the standard phosphate-free LB medium, similarly to the phoD phosphatase isolated from Streptomyces coelicolor [47]. with the activity of the enzyme, which was produced in the standard phosphate-free LB medium, similarly to the phoD phosphatase isolated from Streptomyces coelicolor [47].…”

“…To determine the dependence of CamPhoD activity on the presence of phosphate in the growth medium, the E. coli Rosetta (DE3) cells transformed with the pET40CamPhoD plasmid were grown in 25 mL of the liquid medium containing: bacto-trypton 10 g/L, yeast extract 7.5 g/L, sorbitol 70 g/L, MgCl 2 5 mM, KH 2 PO 4 80 mM, and 25 mg/mL kanamycin at 200 rpm for 16 h at 37 • C [46]. Then, the cells were placed in 1 L of the fresh medium of the abovementioned composition and incubated at 37 • C on a rocking chair at 200 rpm, up to an optical density 0.6-0.8 (λ 600 nm).…”

Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes

“…To construct chimeric plasmid 40HSP70/EIII encoding a hybrid protein containing mature HSP70 and protein EIII, the plasmid 40HSP70 was digested and the gene encoding for EIII was inserted between the SacI/SalI-restriction fragments of the plasmid. Previously developed expression conditions and the medium MX were used for the expression of all plasmids obtained [ 14 ]. Using the method described in [ 14 ], the soluble forms of HSP70 and HSP/EIII were obtained ( Figure 1 ).…”

“…E. coli cells transformed with the recombinant plasmids were grown on a Luria-Bertani (LB) agar (Helicon, Moscow, Russia) plate containing 100 µg/mL kanamycin overnight at 37 °C. A single colony was picked and grown with agitation at 200 rpm in 20 mL of LB medium containing 100 µg/mL of kanamycin at 37 °C overnight, then transferred into 0.5 L of MX medium (Medium for eXpression) [ 14 ]. When the cell density reached an OD 600 of 0.8, 0.2 mM of inducer IPTG was added to the bacterial culture, and the incubation was continued at 18 °C up to 24 h with agitation at 250 rpm.…”