Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities

Li, Yuanli; Long, Guangcheng; Yang, Xiaolan; Hu, Xiaolei; Feng, Yiran; Tan, D.T.S.; Xie, Yanling; Pu, Jun; Liao, Fei

doi:10.1016/j.ijbiomac.2014.12.009

Cited by 12 publications

(2 citation statements)

References 29 publications

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“…For each enzyme/mutant, a individual clone was amplified at 37 °C and 180 rpm, for 12 h with 0.50 mL of the medium in 48-well microplates, but for 4.0 h with 4.0 mL or 250 mL of the medium in glass bottles. Each enzyme/mutant was induced by 1.0 mM isopropyl-β- D -thiogalactoside at 16 °C and 110 rpm for 20 h. Cells were broken by sonication treatment in 20 mM Tris–HCl at pH 8.0; 0.50 mL of the cell lysate after centrifugation at 10,000 rpm for 15 min was filtered through 0.22-μm membrane to serve as a sample for both ITA and activity assay [1] , [2] .…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

et al. 2017

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Data in this article are associated with the research article “Highthroughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins” (Yang et al., 2017) [1]. This article provided data on how to develop an immunoturbidimetric assay (ITA) of enzyme/mutants as proteins in cell lysates in high-throughput (HTP) mode together with HTP assay of their activities to derive their specific activities in cell lysates for comparison, with Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) plus their mutants as models. Data were made publicly available for further analyses.

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Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

et al. 2017

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“…At present, several materials have been introduced to purify His‐tagged protein. Metal‐resin complex is mostly obtained support by fixing metal ions on resin microspheres through complex agents, such as nitrogen triacetic acid (NTA) [10, 11], or iminodiacetic acid (IDA) [12, 13]. However, these materials have some drawbacks like time‐consuming operation, complicated pretreatment, poor mechanical stability [14, 15].…”

Section: Introductionmentioning

confidence: 99%

One‐step selective affinity purification and immobilization of His‐tagged enzyme by recyclable magnetic nanoparticles

Zhou

et al. 2021

Engineering in Life Sciences

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The NiFe2O4 magnetic nanoparticles (NF‐MNPs) were prepared for one‐step selective affinity purification and immobilization of His‐tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier‐transform infrared spectrophotometer and microscopy. The immobilization and purification of His‐tagged GluDH on NF‐MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris‐HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg–1 support, respectively. The immobilized GluDH exhibited high thermostability, pH‐stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni‐NTA resin, the NF‐MNPs displayed a higher specific affinity with His‐tagged recombinant GluDH.

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Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions

Feng

Wang

Liu

et al. 2015

Appl Microbiol Biotechnol

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Bacillus fastidious uricase (BF uricase) containing 322 amino acid residues exhibited high stability under physiological conditions. Its crystal structure was solved to 1.4-Å resolution, showing homotetramer containing two homodimers. After the intersubunit antiparallel β-sheet in its homodimer, each subunit had a total of 18 C-terminus residues forming an α-helix (Q305-A313) and random coil (S314-L322) on surface to bury other two α-helices (I227-T238 and I244-R258). In comparison, reported crystal structures of Arthrobacter globiformis and Aspergillus flavus uricases had atomic coordinates of only some C-terminus residues, while the crystal structures of all the other uricases accessible before September 2014 missed atomic coordinates of all their C-terminus residues, after the intersubunit antiparallel β-sheets. In each homodimer of BF uricase, H-bonds were found between E311 and Y249 and between Y319 and D257; electrostatic interaction networks were found to surround D307 plus R310 and intersubunit R3, K312 plus D257, E318 plus K242, and L322 plus R258. Amino acid mutations that disrupted those interactions when R3 and D307 were reserved caused moderate decreases of activity at pH 9.2 while negligible decreases of activity at pH 7.4, but destroyed stability at pH 7.4 while slightly decreased stability at pH 9.2. Such structural information guided the fusion of 6His-tag to the C-terminus of the mutant L322D with SNSNSN as a linker to reserve the activity and stability. Hence, the folding of the C-terminus residues is crucial for thermal stability of BF uricase under physiological conditions; these new structural insights are valuable for molecular engineering of uricases.

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Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities

Cited by 12 publications

References 29 publications

Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

One‐step selective affinity purification and immobilization of His‐tagged enzyme by recyclable magnetic nanoparticles

Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions

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