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

Golotin, Vasily; Balabanova, Larissa; Noskova, Yu. A.; Slepchenko, Lubov; Bakunina, I. Yu.; Vorobieva, Natalia; Терентьева, Н. А.; Rasskazov, V. A.

doi:10.1016/j.pep.2016.03.006

Cited by 19 publications

(16 citation statements)

References 18 publications

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“…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].…”

Section: Expression Conditions For Camphod Productionmentioning

confidence: 71%

“…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].…”

Section: Expression Conditions For Camphod Productionmentioning

confidence: 99%

“…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).…”

Section: Optimization Of Conditions For Camphod Expressionmentioning

confidence: 99%

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

et al. 2019

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A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein’s subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5′-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5′-pNP-TMP, respectively. The Michaelis–Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S−1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S−1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

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Section: Expression Conditions For Camphod Productionmentioning

confidence: 71%

“…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].…”

Section: Expression Conditions For Camphod Productionmentioning

confidence: 99%

Section: Optimization Of Conditions For Camphod Expressionmentioning

confidence: 99%

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

et al. 2019

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“…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 ).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection.

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Biopharmaceutical applications of α‐galactosidases

Anisha¹

2022

Biotech and App Biochem

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α‐Galactosidases are exoglycosidases that are active on galactose‐containing side chains in oligosaccharides, polysaccharides, glycolipids, and glycoproteins. α‐Galactosidases are gaining increased interest in human medicine, especially in the enzyme replacement therapy for Fabry's disease. α‐Galactosidases with regioselectivity toward α‐1,3‐linked galactose find application in xenotransplantation and blood group transformation. The use of α‐galactosidases as a therapeutic agent in alleviating the postprandial symptoms of irritable bowel syndrome is much acclaimed. The excellent therapeutic applications of α‐galactosidases have led to an upwelling of worldwide research interventions to identify novel α‐galactosidases with improved catalytic efficiency. In addition to these therapeutic applications, α‐galactosidases also have interesting applications in the industrial sectors like food, feed, probiotics, sugar, and paper pulp. The current review focuses on the diverse therapeutic applications of α‐galactosidases and their prospects.

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

Cited by 19 publications

References 18 publications

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

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

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

Biopharmaceutical applications of α‐galactosidases

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