• KSBB Journal
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• v.10 no.5
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• pp.482-490
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• 1995

The relationship between pressure drop and liquid flow rate, for an axial and a radial flow chromatographic column packed with compressible porous media was theoretically analyzed using modified Kozeny-Carman equation. The results were compared with experimental observations obtained using compressible DEAE-agarose as a model medium. At 2-9 psi range studied, the theoretical derivation accounting for 'gel compression' effect predicted simple Langmuirian type response of volumetric flow rate to changes in pressure drop. On the other hand, the experimental response was more or less sigmoidal. At the same pressure drop, radial column showed 2-3 times higher flow rates than those of axial column both theoretically and experimentally. Using r-HBsAg crude extract, protein resolution effects between the two types of columns at different flow rates were compared side-by-side. It turned out that, though general chromatographic behavior was very similar, axial column was somewhat superior in terms of r-HBsAg recovery yield and specificity. However, the number of theoretical plates analysis indicated the protein resolution effects were comparable.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.535-538
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• 2000

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this report, active human leptin was successfully produced in the culture medium of Bacillus subtilis. After simple purification steps consisting of ammonium sulfate precipitation and anion-exchange column chromatography, 2.3 mg of leptin with a purity of greater than 95% was obtained from the 0.5 L culture with the recovery yield of 54.9%. The purified leptin showed the correct folding structure with one disulfide bond.

• BMB Reports
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• v.44 no.4
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• pp.279-284
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• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1751-1757
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• 2007

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.363-380
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• 2018

Wheat is frequently exposed to high temperature during anthesis and ripening period, which resulted in yield loss and detrimental end-use-quality. The transcriptome analysis of wheat under high temperature stress during the early stage of the grain filling period was undertaken. Three expression patterns of differentially expressed genes (DEGs) during grain filling period were identified. The DEGs of seed storage protein and starch-branching enzyme showed continuous increases in their expressions during high temperature stress, as well as during the recovery period. The activities of the enzymes responsible for the elimination of antioxidants were significantly affected by exposure to high temperature stress. Only the WAS-2 family genes showed increased transcription levels under high temperature stress in dehulled spikelets. The relative transcription levels for sub-genome specific WAS-2 genes suggested that WAS-2 genes reacted with over-expression under high temperature stress and decreased back to normal expression during recovery. We propose the role of WAS-2 as a protective mechanism during the stage of grain development under high temperature in spikelets.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.3
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• pp.400-405
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• 2003

The surimi processing from jack mackerel and white croaker muscle using acidic and alkaline solubilization was evaluated. The optimum pH for solubilizing protein in acidic and alkaline range was around 2.5 and 10.5, respectively. The optimum pH value for recovery of protein was around 5. The protein solubility was decreased with increase of salt. The homogenized speed and time for maximum solubility were below 9,500 rpm and 30s, respectively The optimum ratio of water to minced muscle was 6 by evaluating breaking force, deformation and whiteness of cooked gel. The protein yield of alkaline processing is higher than that of conventional processing. In addition, the waste water of conventional processing had high solid, nitrogen content and chemical oxygen demand compare to those of acidic and alkaline processing.

• Journal of Environmental Health Sciences
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• v.31 no.3
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• pp.221-226
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• 2005

Isoflavone was extracted with various concentration of aqueous methanol using whole hypocotyls as the starting material. Whole hypocotyls were preferred as the raw material because the residue could be easily removed from the solvent after the extraction process. Extraction yield was almost constant at the methanol concentration of $20-80\%$. Most of the isoflavone was extracted within 1 hr, and the extraction yield remained almost constant thereafter. When the concentration of methanol was $80\%$, the content of total solid was reduced due to the reduced extraction of contaminating protein as the result of protein insolubilization. Among resins tested, Diaion HP-20, Amberlite XAD-16, and Amberlite IRC-50 showed the highest capacity to absorb the compound. Open column chromatography with Diaion HP-20 showed that $80\%$ aqueous ethanol was most efficient as the eluting solvent with final recovery of the phytochemical being more than $95\%$. Maximum adsorption of the phytochemical occurred at the acidic pH 2-4. When the spatial velocity was increased to 15 and more, the degree of adsorption was decreased, whereas below spatial velocity of 15, the adsorption capacity of isoflavone to the resin was almost constant. The purity of the isoflavone purified by column chromatography was $78\%$.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.51 no.5
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• pp.386-395
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• 2006

The core questions for determining nitrogen topdress rate (Npi) at panicle initiation stage (PIS) are 'how much nitrogen accumulation during the reproductive stage (PNup) is required for the target rice yield or protein content depending on the growth and nitrogen nutrition status at PIS?' and 'how can we diagnose the growth and nitrogen nutrition status easily at real time basis?'. To address these questions, two years experiments from 2001 to 2002 were done under various rates of basal, tillering, and panicle nitrogen fertilizer by employing a rice cultivar, Hwaseongbyeo. The response of grain yield and milled-rice protein content was quantified in relation to RVIgreen (green ratio vegetation index) and SPAD reading measured around PIS as indirect estimators for growth and nitrogen nutrition status, the regression models were formulated to predict PNup based on the growth and nitrogen nutrition status and Npi at PIS. Grain yield showed quadratic response to PNup, RVIgreen around PIS, and SPAD reading around PIS. The regression models to predict grain yield had a high determination coefficient of above 0.95. PNup for the maximum grain yield was estimated to be 9 to 13.5 kgN/10a within the range of RVIgreen around PIS of this experiment. decreasing with increasing RVIgreen and also to be 10 to 11 kgN/10a regardless of SPAD readings around PIS. At these PNup's the protein content of milled rice was estimated to rise above 9% that might degrade eating quality seriously Milled-rice protein content showed curve-linear increase with the increase of PNup, RVIgreen around PIS, and SPAD reading around PIS. The regression models to predict protein content had a high determination coefficient of above 0.91. PNup to control the milled-rice protein content below 7% was estimated as 6 to 8 kgN/10a within the range of RVIgreen and SPAD reading of this experiment, showing much lower values than those for the maximum grain yield. The recovery of the Npi applied at PIS ranged from 53 to 83%, increasing with the increased growth amount while decreasing with the increasing Npi. The natural nitrogen supply from PIS to harvest ranged from 2.5 to 4 kg/10a, showing quadratic relationship with the shoot dry weight or shoot nitrogen content at PIS. The regression models to estimate PNup was formulated using Npi and anyone of RVIgreen, shoot dry weight, and shoot nitrogen content at PIS as predictor variables. These models showed good fitness with determination coefficients of 0.86 to 0.95 The prescription method based on the above models predicting grain yield, protein content and PNup and its constraints were discussed.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.