• Maxillofacial Plastic and Reconstructive Surgery
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• v.19 no.3
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• pp.265-286
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• 1997

Bone morphogenetic proteins(BMPs) are a group of transforming growth factor beta(TGF-${\beta}$)-related factors and multifunctional proteins, especially the only known biologic factors capable of inducing endochondral bone formation at an extraskeletal site. This study was performed to investigate the effect of the partially purified porcine BMP(pBMP) at an ectopic site. PBMP was partially purified from porcine bone matrix and its activity was monitored by an in vivo bioassay. The purification method utilized extraction of the bone-inducing activity with 4M guanidine, followed by chromatography on heparin-Sepharose. Active fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. And the fractions were reconstituted with inactive insoluble collagenous bone matrix from rats, acid soluble type I collagen from rat tail and chondroitin-6-sulfate sodium salt and implanted into the pectroralis muscle pouches of Sprague-Dawley rats. And the carrier complex was implanted on the opposite side as control. The rats were sacrificed at the day of 1st, 3rd, 5th, 7th, 11th, 14th and 21st after implantation and examined histologically, radiologically and biochemically. And alkaline phosphatase activity and calcium content were used as indices of bone formation. The results were as follows ; 1. Active fractions were localized in a zone between 31 and 40 KDa on SDS-PAGE. 2. The implanted 3.0mg of the partially purified pBMP induced cartilage and bone in the muscle tissue of rats through an endochondral ossification process. 3. Inactive insoluble bone matrix, type I collagen and chondroitin-6-sulfate have functioned as carriers for pBMP, but revealed some foreign body reactions. 4. Soft X-ray didn't reveal significant change between the experimental and the control group. 5. The alkaline phosphatase activities in the experimental group of 5th, 7th, 11th, 14th and 21st were increased significantly compared with control (p<0.01) with the peak in the group of 11th day. 6. With time, the calcium content of the experimental group increased. And the calcium contents in the experimental group of 11th, 14th and 21st were increased significantly compared with control (p<0.01).

• Journal of the Korean Society of Food Science and Nutrition
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• v.15 no.3
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• pp.276-285
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• 1986

This study was undertaken to investigate the characteristics of the proteolytic enzyme extracted from Neungee mushroom [Sarcodon aspratus (Berk.) S. Ito]. The enzyme was purified by using Tris-acryl CM-cellulose ion exchange, gel filtration on Ultrogel AcA 54, Hydroxy apatite column chromatography and preparative isoelectic focusing. The specific activity of the purified enzyme increased 8 times as compared with that of the crude enzyme. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE). The optimum pH was 10.1, indicating the enzyme to be alkaline protease and the optimum temperature was $57^{\circ}C$. The enzyme was stable at temperatures lower than $50^{\circ}C$and at pH values ranging from 4.0 to 10.8. However, the enzyme activity decreased by 26 and 65% at 60 and $65^{\circ}C$, respectively, when incubated for 30 minutes. The enzyme activity was activated by $Mn^{++}$ and inhibited by $Cu^{++}$ and $Hg^{++}$. The enzyme was consisted of monomer and its molecular weight estimated to be about 30,100 when determined by sodium dodecyl sulfate PAGE. Isoelectric point of the enzyme was determined to be 9.80.

• Journal of the Korean Chemical Society
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• v.39 no.6
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• pp.459-465
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• 1995

Acetolactate Synthase (ALS) was partially purified from the yeast and its basic biochemical studies were carried out. Yeast was grown in the minimum media containing 0.5% glucose, 51 mM $K_2HPO_4$, 22 mM $KH_2PO_4$, 8 mM $(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$ for 18 hours at 37 $^{\circ}C$. The cell was ruptured in the buffer (20 mM phosphate buffer pH 7.0, 0.1 mM TPP, 0.5 mM DTT, 1 ${\mu}M$ FAD, and 1 mM MgCl_2$) following an overnight suspension. The supernatant fraction was collected from $10,000{\times}g$ and the enzyme was further purified by ammonium sulfate fractionation, DEAE-Sephacel chromatography and leucine-agarose chromatography. The enzyme activity was measured under the various conditions by the function of protein concentration, time, temperature, pH, and substrate. The optimum temperature was found to be 50$^{\circ}C$, optimum pH 8.0∼8.5. The kinetic parameters, $K_m\;and\;V_{max}$ were 8.4 mM and 17.9 nmol/mg/min respectively. Stability of the enzyme was studied with ethylene glycol and glycerol added to the enzyme solution. Both ethylene glycol and glycerol improved the enzyme stability up to 50%. The study of feedback inhibition showed that valine was a strong inhibitor while leucine was a weak inhibitor.

• The Korean Journal of Zoology
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• v.28 no.1
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• pp.31-43
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• 1985

The $(Ca^{2+}+Mg^{2+})$-ATPase has been purified homogeneously from sarcoplasmic reticulum of rat skeletal muscle by sucrose density gradient centrifugation. The purified enzyme has a molecular weight of 115,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dedecyl sulfate, and therefore has the same size of the enzyme in rabbit and chick skeletal muscle. $Ca^{2+}, Mg^{2+}, Fe^{2+}, Co^{2+}, and Mn^{2+}$ at 50 $\\muM$ show stimulatory effect on the ATP-ase, while $Zn^{2+}, Cu^{2+}, and Hg^{2+}$ inhibit it at the same concentration. The ATPase activity is insensitive to antimalarial drugs such as quinine and quinacrine, but is sensitive to inhibition by p-hydroxymecurie benzoate and phenylmethylsulfonylfluoride. The enzyme has optimum pH of 6 to 7 and Km value for ATP is estimated to be 98 $\\muM$. Thus, a number of biochemical properties of this enzyme appear to be different from those of the enzyme that have been isolated from rabbit skeletal muscle. The $(Ca^{2+}+Mg^{2+})$-ATPase appears to be selectively degraded in microsomal fraction. The activity of metalloendoprotease is evident in the microsomal preparation when assayed by radioactively labeled protein substrate, such as $^{3}H-casein and $^{125}I$-insulin. However, it is presently unclear whether the metalloendoprotease is responsible for the degradation of the $(Ca^{2+}+Mg^{2+})$-ATPase.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• The Korean Journal of Physiology
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• v.24 no.2
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• pp.363-375
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• 1990

The present investigation was performed to purify bombesin-like immunoreactivity (BBS-LI) from the skin of frogs, B. orientalis inhabiting Korea. For extraction of BBS-LI, the fresh skin of 360 g from frogs was immersed in 1,800 ml of 100% methanol and then kept at $4^{\circ}C$ for 5 days. BBS-LI was partially purified by liquid chromatography using an alkaline alumina column followed by a Sephadex G-10 column. BBS-LI was further purified by using sequential HPLC of reversed phase C18 preparation, gel permeation, SP-ion exchange and reversed phase C18 analysis. BBS-LI in fractions of each step was monitored by radioimmunoassay for which bombesin antiserum with a titer of 1 : 188,800 was raised in a guinea pig. Eventually, two different BBS-LI were successfully purified and each BBS-LI showed the following character. 1) BBS-LI was well separated into two peaks in SP-ion exchange HPLC. One (BBS-LI-K1) bound to the column while the other (BBS-LI-K2) did not. 2) BBS-LI-K1, 73.8% of total BBS-LI, was not differentiated from synthetic bombesin in reversed phase C18 analytical and gel permeation HPLC. 3) BBS-LI-K2, 26.2% of total BBS-LI, eluted later than synthetic bombesin in reversed phase C18 analytical HPLC, but it eluted with a retention time identical to that of synthetic bombesin in gel permeation HPLC. 4) The two forms of BBS-LI and synthetic bombesin identically stimulated gastrin release and pancreatic exocrine secretion including volume, protein output and amylase output in anesthetized rats. It is concluded from the above results that the skin of B. orientalis contains two different forms of BBS-LI which are very identical to bombesin immunologically and biologically. In comparison with synthetic bombesin containing 14 amino acid residues, the major form shows quite similar pattern in all HPLC used in the present study, but the minor form exhibits quite different pattern in SP-ion exchange and reversed phase C18 analytical HPLG.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.145-149
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• 2012

Cecropin is a well-studied antimicrobial peptide that play important role as key factor in insect humoral immunity. In this study, cecropin-like antimicrobial peptide was isolated and purified from the larval haemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. To isolate antimicrobial peptide, we separated and compared acidic extracted hemolymph protein bends between control and immune-challenged larvae using SDS-PAGE analysis. In the immune hemolymph extract, but not of non-immune hemolymph, we detected differential expressed peptide band with molecular mass 4,223.01 Da. To understand this peptide better, we successfully purified this peptide using cation exchange chromatography and gel permeation chromatography. Its N-terminal amino acid sequence obtained by Edman degradation evidenced a significant degree of identity with other lepidopteran cecropins. The purified A. yamamai cecropin-like peptide showed a broad spectrum of activity against fungi, Gram-negative and Gram-positive bacteria.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.5 no.1
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• pp.18-25
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• 2019

2-Nitroimidazole derivatives have been reported to accumulate in hypoxic tissue. We prepared a novel $^{99m}Tc-MAG_3$-2-nitroimidazole and evaluated the feasibility for hypoxia imaging agent. $Bz-MAG_3$-2-nitroimidazole was synthesized by direct coupling of $Bz-MAG_3$ and 2-nitroimidazole using dicyclohexylcarbodiimide. $Bz-MAG_3$-2-nitroimidazole was labeled with $^{99m}Tc$ in the presence of tartaric acid and $SnCl_2-2H_2O$ at $100^{\circ}C$ for 30 min. And the reaction mixture was purified by $C_{18}$ Sep-pak cartridge. The labeling efficiency and the radiochemical purity were checked by ITLC-SG/acetonitrile. The tumor was grown in balb/c mice for 8~13 days after the subcutaneous injection of tumor cells, CT-26 (murine colon adenocarcinoma cell). Biodistribution study and tumor autoradiography were performed in the xenografted mice after i.v injection of 74 kBq/0.1 mL and 19 MBq/0.1 mL of $^{99m}Tc-MAG_3$-2-nitroimidazole, respectively. In vivo images of $^{99m}Tc-MAG_3$-2-nitroimidazole in tumor bearing mice were obtained 1.5 hr post injection. The labeling efficiency was $45{\pm}20%$ and the radiochemical purity after purification was over 95%. Paper electrophoresis confirmed negative charge of $^{99m}Tc-MAG_3$-2-nitroimidazole. $^{99m}Tc-MAG_3$-2-nitroimidazole was very stable at room temperature and its protein binding was 53%. The $^{99m}Tc-MAG_3$-2-nitroimidazole exhibited high uptake in the liver, stomach and intestine. In biodistribution study using tumor bearing mice, the uptakes (% ID/g) of the tumor were $0.5{\pm}0.1$, $0.4{\pm}0.0$, $0.2{\pm}0.1$ and $0.1{\pm}0.1$ at 5, 15, 30 min and 4 hrs. Tumor/muscle ratio were $1.4{\pm}0.1$, $2.2{\pm}0.83$, $3.0{\pm}0.9$, and 3.7 (n=2) for 5, 15, 30 min and 4 hrs. The uptake in hypoxic area was found higher than in non-hypoxic area of tumor tissue by autoradiography. In vivo images showed the relatively faint uptake to the hypoxic tumor region. $^{99m}Tc-MAG_3$-2-nitroimidazole was successfully synthesized and found feasible for imaging hypoxia.

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.329-336
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• 2021

Cellulophga sp. J9-3, is a gram-negative, aerobic marine bacterium belonging to the family Flavobacteriaceae. In addition to cellulose degradability, the J9-3 strain is also capable of hydrolyzing agar in the solid and liquid medium, and the production of agarase in the presence of agarose can be remarkably induced by the bacterium. From the cell culture broth of Cellulophga sp. J9-3, ammonium sulfate precipitation and three kinds of column chromatography were successively performed to purify a specific agarase protein, the AgaJ93. Purified AgaJ93 showed the strongest hydrolyzing activity towards agarose (approximately 22%), and even displayed activity towards starch. AgaJ93 hydrolyzed agarose into neoagarotetraose and neoagarohexaose via various oligosaccharide intermediates, indicating that AgaJ93 is an endo-type β-agarase. AgaJ93 showed maximum activity at a pH of 7.0 and temperature of 35 ℃. Its activity increased by more than six times in the presence of Co²⁺ ions. The N-terminal sequence of AgaJ93 showed 82% homology with the heat-resistant endo-type β-agarase Aga2 of Cellulophaga sp. W5C. However, the biochemical properties of the two enzymes were different. Therefore, AgaJ93 is expected to be a novel agarose, different from the previously reported β-agarases.