Modification of C-segment of Cfa DnaE split intein for improving clean-in-place in chromatography process.

This research focuses on the construction of an affinity purification system based on Cfa DnaE split intein. Cfa DnaE intein is an artificially constructed intein with the advantages of a fast cleavage reaction and good stability. In a previous study, a purification system that uses Cfa intein as a tag was constructed, the separation of the target protein and the tag during the purification process was completed, and the purity of the purified target protein reached 98.21%. Guided by molecular docking results, we identified flexible regions in the split intein and inserted several glycines into the protein to decrease the stability of the Cfa IC , thereby improving the regenerability of the IN media. Inserting 6 glycines between amino acids 14 and 15 of IC improved the regeneration rate of IC -GFP on the column to approximately 96%.

Flexible FeVOx porous nanorods on carbon cloth for long-life aqueous energy storage.

We report the FeVOx porous nanorods on carbon cloth as a novel cathode material for flexible aqueous energy storage. It exhibits excellent electrochemical properties and cycling stability in supercapacitors and zinc-ion batteries. Moreover, this work makes significant progress for developing high-performance electrodes and provides a foundation for future research.

Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein.

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0 mg mL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.

Corallincola holothuriorum sp. nov., a facultative anaerobe isolated from sea cucumber intestine.

Strain C4T, isolated from sea cucumber intestine in Weihai, Shandong, PR China, is a novel Gram-stain-negative, facultatively anaerobic, amphitrichously flagellated, short rod that grows as creamy white bacterial colonies on plates. Optimal growth of the strain was observed at 28-30 °C, pH 6.5-7.0 and at a concentration of 3 % NaCl. The G+C content of the genomic DNA was 49.0 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain C4T is a member of the genus Corallincola and was most similar to Corallincola platygyrae JLT2006T. The major cellular fatty acids of strain C4T were C16 : 1ω7c/iso-C15 : 0 2-OH, C16 : 0 and C18 : 1ω7c. The sole respiratory quinone was Q-8. The predominant polar lipids in strain C4T were phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. Based on morphology and physiological characteristics, strain C4T should be classified as a novel species in the genus Corallincola, for which Corallincolaholothuriorum is proposed. The type strain is C4T (=ATCC BAA-2611T=CICC 10839T).

Bizionia sediminis sp. nov., isolated from coastal sediment.

A Gram-stain-negative, aerobic, non-gliding, rod-shaped and orange-coloured bacterium, designated strain P131T, was isolated from marine sediment of the coast of Weihai, China, and subjected to a polyphasic study. Strain P131T was found to grow optimally at 28-30 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain P131T was found to belong to the genus Bizionia and exhibited 94.6-97.0 % 16S rRNA gene sequence similarity with recognized Bizionia species. The dominant cellular fatty acids of strain P131T were identified as iso-C15 : 0, iso-C15 : 0 G, iso-C17 : 0 3-OH and iso-C17 : 1ω9c. The predominant polar lipids were phosphatidylethanolamine, phospholipid, two aminolipids and two unidentified lipids. The predominant respiratory quinone was menaquinone MK-6 and the DNA G+C content was 36.7 mol%. On the basis of the phylogenetic and phenotypic evidence presented, strain P131T represents a novel species of the genus Bizionia, for which the name Bizionia sediminis sp. nov. is proposed. The type strain is P131T (=KCTC 42587T=MCCC 1H00124T).

Aquimarina rubra sp. nov., isolated from sediment of a sea cucumber culture pond.

A Gram-stain-negative, non-motile, rod-shaped, red-pigmented, facultatively anaerobic bacterium, designated SS2-9T, was isolated from sediment collected from a sea cucumber culture pond located in Rongcheng, Shandong province, China. Cells of strain SS2-9T were approximately 0.3-0.5 µm in width and 1.5-6.0 µm in length. The strain was able to grow at 10-37 °C, at pH 6.5-8.5 and in the presence of 0.5-6.0 % (w/v) NaCl. It grew optimally at 28 °C and in the presence of 2.0 % (w/v) NaCl. The DNA G+C content was 34.5 mol% and the sole respiratory quinone was menaquinone 6 (MK-6). The predominant cellular fatty acids were C15 : 0, iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified phospholipid, two unidentified aminolipids and four unidentified lipids. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SS2-9T was phylogenetically related to members of the genus Aquimarina and was closely related to Aquimarina amphilecti 92VT (97.29 % similarity). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain SS2-9T was considered to represent a novel species of the genus Aquimarina, for which the name Aquimarina rubra sp. nov. is proposed. The type strain is SS2-9T (=KCTC 52274T=MCCC 1H00142T).

Mixed-mode chromatography integrated with high-performance liquid chromatography for protein analysis and separation: Using bovine serum albumin and lysozyme as the model target.

A type of mixed-mode chromatography was integrated with high-performance liquid chromatography for protein analysis and separation. The chromatographic behavior was tested using bovine serum albumin and lysozyme as model proteins. For the mixed-mode column, the silica beads were activated with γ-(2,3-epoxypropoxy)-propytrimethoxysilane and coupled with 4-mercaptopyridine as the functional ligand. The effects of pH, salt, and the organic solvent conditions of the mobile phase on the retention behavior were studied, which provided valuable clues for separation strategy. When eluted with a suitable pH gradient, salt concentration gradient, and acetonitrile content gradient, the separation behavior of bovine serum albumin and lysozyme could be controlled by altering the conditions of the mobile phase. The results indicated this type of chromatography might be a useful method for protein analysis and separation.

Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography.

Protein A of Staphylococcus aureus has been widely used as an affinity ligand for the purification of immunoglobulin. However, the low elution pH and the sensitivity to alkaline condition restricted the large-scale application of antibody purification. To overcome these disadvantages, the B domain was selected and mutated to Z domain and the recombinant Protein A was reconstructed by linking five Z domains. First, a section of six glycines was inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.0-5.0. Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work confirmed the modification of Protein A and exhibited the characteristics of recombinant Staphylococcal Protein A for antibody purification.

Salegentibacter echinorum sp. nov., isolated from the sea urchin Hemicentrotus pulcherrimus.

A novel Gram-negative, strictly aerobic, heterotrophic, non-motile and yellow-pigmented bacterial strain, designated HD4(T), was isolated from the sea urchin Hemicentrotus pulcherrimus collected from the Yellow Sea in China. Optimal growth of the strain was observed at 28-30 °C, pH 6.8-7.3, and in the presence of 3-5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain HD4(T) exhibited high similarity with the members of Salegentibacter (92.3-95.4 %). The DNA G+C content was 37.0 mol%, MK-6 was the main respiratory quinone and summed feature 3 (comprising iso-C15:0 2-OH/C16:1ω7c), iso-C15:0, iso-C17:0 3-OH and anteiso-C15:0 were the major cellular fatty acids. The predominant polar lipids in strain HD4(T) were phosphatidylethanolamine and two unknown lipids (L2, L4). Based on the phylogenetic, physiological and biochemical characteristics, strain HD4(T) should be classified as a novel species within the genus Salegentibacter, for which the name Salegentibacter echinorum sp. nov. is proposed. The type strain is HD4(T) (=CICC 10466(T) = NRRL B-59666(T)).

[Cloning and functional analysis of SCTF-1 encoding a C2H2-type Zinc finger protein from soybean].

The zinc finger protein is one of the proteins with finger-like domain. Some of them are transcription factors which play important role in plant growth and plant resistance to abiotic stresses. In this paper, a novel C2H2-type zinc finger protein gene SCTF-1 (GenBank accession number JQ692081) was isolated from soybean (Glycine max (L.) Merr.) This gene has a 699 bp ORF (open reading frame) with no intron and encodes a 24.9 kDa protein with 233 amino acids. Its isoelectric point (pI) is 8.33. The SCTF-1 protein contains two typical C2H2-type zinc finger domains. Both of them have highly conserved amino acid sequence-QALGGH which is a particular characteristic of plant. Transient expression of the GFP-SCTF-1 protein in onion epidermal cell showed that SCTF-1 was localized in cell nuclei. RT-PCR results showed that SCTF-1 gene was expressed with high levels in flowers and leaves in soybean, but low in roots and stems. The expression of SCTF-1 gene was strongly induced by low temperature in the soybean seedlings. Overexpression of SCTF-1 enhanced cold tolerance of transgenic tobacco (Nicotiana tabacum L.) compared to the control.

Chromatographic performance of macroporous cellulose-tungsten carbide composite beads as anion-exchanger for expanded bed adsorption at high fluid velocity.

Macroporous cellulose-tungsten carbide composite beads was designed and prepared as an anion-exchanger for expanded bed adsorption (EBA). The wet density of composite beads was adjusted at the range of 1.2-2.4 g/ml with the control of tungsten carbide addition, and optimized for EBA at high fluid velocity. The results indicated that the wet density of composite beads could increase linearly with the increase of tungsten carbide addition, meanwhile other physical properties, such as size, porosity, specific surface area, mean pore diameter, etc., were hardly or slightly influenced. The composite beads were coupled with diethylaminoethyl (DEAE) as an anion-exchanger for EBA. The expansion characteristics in expanded bed were investigated and sensitively changed as the wet density of composite beads, corresponding to tungsten carbide addition in the preparation. The relation among the operation fluid velocity, the ratio of tungsten carbide to cellulose viscose in the preparation and the expansion factor was found, which could be used to predict the operation velocity of composite beads with varying tungsten carbide addition. The liquid mixing in expanded bed was also tested and showed good bed stability for EBA processes. With the adsorption equilibrium experiments, the saturated adsorption capacity of bovine serum albumin could reach 68.7 mg/g adsorbents (equal to 97.1 mg/ml adsorbents). The ratio of Q(10%) (the dynamic adsorption at 10% breakthrough) in expanded bed to packed bed could reach more than 90% for the fluid velocity of 500 cm/h, even 77.1% for the fluid velocity as high as 900 cm/h. The chromatographic results demonstrated that the composite beads prepared are suitable for EBA applications at high fluid velocity.

Preparation and characterization of macroporous cellulose-tungsten carbide composite beads for expanded bed applications.

The specially designed adsorbent is one of the most important bases to achieve the expanded bed adsorption (EBA) process. The perfect adsorbents tend to be of small size, large pore and high density. In the present work a new kind of macroporous cellulose beads densified by high-density tungsten carbide were prepared through the method of water-in-oil suspension thermal regeneration. The cassava starch was used as porogenic agent during the preparation. Firstly the gelatinized starch solution was added into the cellulose viscose. After the formation of cellulose beads, the composite starch could be eliminated with boiling water wash and enzymatic hydrolyzation. The sphericity of cellulose beads was not influenced by starch addition and some amount of large pores with the diameter of about 1-3 microm could be formed in the beads. Varieties of physical properties, such as wet density, water content, porosity, specific surface area and mean pore diameter of the composite beads, as the function of starch addition were investigated. The analysis of elution peaks with model proteins (packed bed mode) indicated that the mass transfer kinetic inside the macroporous beads prepared was enhanced, which might certainly benefit the chromatographic operation under high flow rate. In addition, the prepared beads showed good expansion and stability in expanded bed, and are thus suitable for expanded bed applications.

Evaluation of new high-density ion exchange adsorbents for expanded bed adsorption chromatography.

New adsorbents Q HyperZ and CM HyperZ composed of hydrogel-filled porous zirconium oxide particles were evaluated for expanded bed adsorption applications in the present work. The HyperZ adsorbents have wet density of 3.16 g ml(-1), particle size of 44.5-100.8 microm and average sphere diameter of 67 microm. The bed expansion as the function of flow velocity and fluid viscosity was measured and correlated with Richardson-Zaki equation. The suitable expansion factor was considered less than 2.5, while the corresponding flow velocity was about 450 cmh(-1). Liquid mixing in the bed was determined to evaluate the stability of expanded bed. The Bodenstein numbers tested were higher than 40 and the axial mixing coefficients (D(ax)) were between 0.5 and 9.7x10(-6)m(2)s(-1), which demonstrated that a stable expanded bed could be formed under suitable operation conditions. Bovine serum albumin (BSA) and lysozyme were used as model proteins to estimate the adsorption capacities of Q and CM HyperZ, respectively. The maximum equilibrium adsorption of Q and CM HyperZ could reach 45.7 and 27.2 mg g(-1) drained adsorbents, respectively. It was found that yeast cells had little influence on the adsorption capacities of the two adsorbents tested. The dynamic adsorption capacity of BSA at 10% breakthrough with Q HyperZ was 35.9 mg g(-1) drained adsorbent at flow velocity of 100 cm h(-1) for packed bed adsorption. The values for expanded bed adsorption were 34.4 mg g(-1) drained adsorbent at flow velocity of 200 cm h(-1), 33.6 mg g(-1) drained adsorbent at 300 cm h(-1) and 31.7 mg g(-1) drained adsorbent 400 cm h(-1). The results demonstrated that Q HyperZ and CM HyperZ are suitable for expanded bed adsorption of biomolecules.