• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1742-1750
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• 2015

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at $15^{\circ}C$, whereas the reaction at $25^{\circ}C$ was faster than that at $15^{\circ}C$. The refolding yield at $15^{\circ}C$ was 17% higher than that at $25^{\circ}C$. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at $15^{\circ}C$ for 16 h. The maximum refolding yield was 74.8% at $15^{\circ}C$ with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.

• Archives of Plastic Surgery
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• 제34권1호
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• pp.13-17
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• 2007

Purpose: To understand the pathogenesis of the disease that presents abnormally proliferated vascular endothelial cells, a model of DMH(1,2-dimethylhydrazine)-induced abnormal proliferation of HUVECs(Human Umbilical Vein Endothelial Cells) was made. We indirectly determined that Protein Kinase C(PKC) restricts the cellular proliferation and inhibits the manifestation of growth factor by using several inhibiting substances of the transmitter through our previous studies. Thereupon, we attempted to observe direct enzymatic activities of PKC and its correlation with the abnormal proliferation of vascular endothelial cells. Methods: $10^5$ HUVECs cells were applied to 6 individual well plates in three different groups; A control group cultured without treatment, a group concentrated with $0.75{\times}10^{-8}M$ DMH only, and a group treated with DMH & $5{\times}10^{-9}M$ Calphostin C, inhibitor of PKC. In analyzing the formation of intracellular PKC enzyme, protein separation was performed, and separated protein was quantitatively measured. PKC enzyme reaction was analyzed through Protein Kinase C Assay System (Promega, USA), and the results were analyzed according to Beer's law. Results: Enzymatic activity of PKC presented the highest in all reaction time of a group concentrated only with DMH, and the lowest in the control group. The group treated with DMH and the inhibitor revealed statistically lower enzymatic activity than group only with DMH in all reaction time, although higher than the control group. Conclusion: From the enzymatic aspect, most active and immediate reaction of the PKC was observed in the group concentrated with DMH only. The group treated with DMH & PKC inhibitor showed meaningful decrease. Accordingly, PKC holds a significant role in DMH-induced abnormal proliferation of vascular endothelial cells.

• 대한미생물학회지
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• 제21권1호
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• pp.145-149
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• 1986

The development of delayed-type hypersensitivity(DTH) reaction to Staphylococcus aureus in mice was studied, Mice received 3 injections of $10^8$ viable S. aureus subcutaneously showed a marked footpad swelling when mice were challenged with $10\;{\mu}g$ staphylococcal protein antigen into footpad(The percent increase of footpad thickness at 24 h after challenge wsa 35% approximately). Histological observation of footpad of immunized mice showed a marked thickness of subcutaneous tissue due to edematous reaction and massive infiltration of lymphocytes and neutrophils which are characteristic cells in DTH reaction. Intensity of DTH reaction of mice immunized with viable bacteria was much higher than that of mice immunized with staphylococcal protein or heat-killed bacteria. The DTH reaction to S. aureus could be transferred to normal recipient mice by both spleen cells and lymph node cells.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.75-80
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• 2000

An improved method of refolding recombinant human proinsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured proinsulin-s-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 50 l at protein concentration of 1 mg/ml. The optimum urea concentration was 1M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5mg/ml was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore, making this process economical.

• 생명과학회지
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• 제14권1호
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• pp.17-20
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• 2004

대장균 트립토판 합성효소의 $\beta$반응의 빠른 속도 반응에 일가 양이온의 영향을 D56N과 D56G 잔기치환체를 대상으로 조사하였다. 일가 양이온을 처리하였을 때 생성되는 중간반응 생성물의 생성과 분해 속도가 영향을 받았다. 56번 자리 잔기치환체도 야생형에 비해 반응의 중간산물의 생성과 분해에 있어 모두 느린 속도를 보여주고 있다. 이들 잔기 치환체에 미치는 일가 양이온의 영향 또한 달라졌다. 이 결과는 대장균의 효소에서도 56번잔기가 이소조절에 관여하고 있고, 이 과정에서 일가 양이온들이 이소조절 리간드역할을 수행하는 것을 보여준다.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제4권2호
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• pp.238-242
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• 2001

저자들은 우유 단백질에 대한 FPIES의 1례를 경험하였기에 문헌 고찰과 함께 보고하는 바이다.

• 한국수산과학회지
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• 제30권6호
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• pp.948-955
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• 1997

Immunomagnetic separation of virus coupled with .reverse transcription-polymerase chain reaction (IMS-PCR) was performed with infectious hematopoietic necrosis virus (IHNV). A DNA fragment of expected size was synthesized in the RT-PCR with total RNA extracted from IHNV inoculated CHSE-214. In a SDS-PAGE analysis, a protein band of over 70kDa was detected from non-infected cells and cells inoculated with IHNV and infectious pancreatic necrosis virus (IPNV). This protein was detected in the Western blot analysis probably because of non-specific reaction to monoclonal antibody against IHNV nucleocapsid protein. In the immunomagnetic separation, magnetic beads coated with monoclonal antibody against the IHNV nucleocapsid protein was incubated with supernatant from IHNV inoculated CHSE-214 cells. During this process, the non-specifically reacting protein could be removed by washing the magnetic bead with PBS in the presence of an external magnetic field, and viral proteins were detected from the remaining, cleaned magnetic beads. It was necessary to extract viral RNA from the captured virus particles before RT-PCR, and no DNA product was detected when the captured virus was only heated 5 min at $95^{\circ}C$. A PCR-product of expected size was synthesized from IMS-PCR with magnetic beads double coated either by goat anti-mouse IgG antibody -monoclonal antibody or streptavidin - biotin conjugated monoclonal antibody.

• Biomaterials and Biomechanics in Bioengineering
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• 제3권1호
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• pp.59-69
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• 2016

A surface resisting protein adsorption and cell adhesion is highly desirable for many biomedical applications such as diagnostic devices, biosensors and blood-contacting devices. In this study, a surface conjugated with sulfobetaine molecules was fabricated via the click reaction for the anti-fouling purpose. An alkyne-containing substrate (Alkyne-PPX) was generated by chemical vapor deposition of 4-ethynyl-[2,2]paracyclophane. Azide-ended mono-sulfobetaine molecules were synthesized and then conjugated on Alkyne-PPX via the click reaction. The protein adsorption from 10% serum was reduced by 57%, while the attachment of L929 cells was reduced by 83% onto the sulfobetaine-PPX surface compared to the protein adsorption and cell adhesion on Alkyne-PPX. In conclusion, we demonstrate that conjugation of mono-sulfobetaine molecules via the click chemistry is an effective way for reduction of non-specific protein adsorption and cell attachment.

• Environmental Sciences Bulletin of The Korean Environmental Sciences Society
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• 제3권3호
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• pp.177-188
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• 1999

The effect of ABA on the chloroplast disassembly of Zea mays was investigated by measuring the changes in the relative distribution of chlorophyll(Chl) between the Chl-protein complexes in ABA treated and untreated sensecting leaves. The reaction center(RC)-light harvesting complex(LHC) regions were rapidly disassembled in the late stage of dark-induced senescence. Plus, during dark-induced senescence, the disassembly of a reaction center of P700 apoproteins containing mainly Chl a was faster than that of a reaction center of LHCI apoproteins containing both Chl a and Chl b. The increase in the relative distribution of Chl-protein complexes in the RC-Core2 in the late stage of senescence was due to the accumulation of core complexes such as CP47/43 and reaction centers including D1/D2 apoproteins disassembled from the RC-Corel containing the dimer of D1/D2 apoproteins. The LHCII region was more stable than the other Chl-protein complexes throughout leaf senscence. Accordingly, it is suggested that the preferential breakdown of Chl a gives rise to the disassembly of Chl a-binding proteins, particularly reaction centers and core complexes during dark-induced senescence, plus the primary target of the photosynthetic apparatus in sensecing leaves would seem to be Chl a along with the proteins associated with Chl a. The application of ABA promoted the disassembly of the P700 apoproteins in the PSI reaction center and the dimer of D1/D2 apoproteins, and the conversion of the trimeric LHCII apoprotein to the monometirc LHCII apoprotein during the middle stage of leaf senescence, thereby suggesting that ABA accelerates the disassembly of both Chl a-binding and Chl a+b-binding proteins, particularly Chl a-binding proteins during the middle stage of leaf senescence.

• 대한수의학회지
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• 제43권3호
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• pp.507-516
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• 2003

This study was performed to clarify the induction possibility of anaphylactoid reactions by the administration with the heartworm extracts, and, if any, to elucidate different virulences in terms of the protein concentrdtions and sexes of Dirofilaria immitis. Twenty three clinically healthy D. immitis-free adult dogs were used in the present study. The experimental animals were divided into 5 groups. Group A (5 heads) was administered with an female heartworm extract containing 0.1 g/dl protein concentration. Group B (4 heads) was administered with an male heartworm extract containing 0.1g/dl protein concentration. Group C (5 heads) was administered with an female heartworm extract containing 0.2 g/dl protein concentration. Group D (4 heads) was administered with an male heartworm extract containing 0.2 g/dl protein concentration. Group E (5 heads) was administered with an female heartworm extract containing 0.4 g/dl protein concentration. The changes of clinical symptoms and vital signs (body temperature, heart rate and respiration rate) were examined before and 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours and 24 hours after injection with the extracts, respectively. In addition, the changes of hematological values (RBC, PCV and total leukocytes counts), serum chemical values (ALP and CK) were determined. It was considered that heartworm extract could induce anaphylactoid reaction and adult female heartworm extract was more affective than those of adult male heartworm extract in the changes of clinical symptoms, vital signs, hematological values and serum chemical values.