• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.60 no.1
• /
• pp.33-40
• /
• 2015

Soybean is very useful crop to supply vegetable protein for human. Supply of soybean is increased because it has useful ingredient. Recently, cultivation of soybean in paddy field is increasing due to the increase of rice stockpile in Korea. Hence, in this study, expression of protein was identified regarding different environment for cultivation to investigate the effect of different environment on protein expression. Two-dimensional electrophoresis was performed to investigate the expression of protein using image analysis program to measure degree of protein expression in numerical value. Hannam-kong, Beakcheon-Kong, Hwangkeum-Kong, and Danwon-Kong were used as plant material. 2-DE combined with image analysis revealed that each degree of protein expression of Hannam-Kong and Hwangkeum-Kong in upland field was higher than degree of protein expression in paddy field. However, in case of Beackcheon-Kong, the phenomenon was opposite. In Danwon-kong, the degree of protein expression was not different between up-land field and paddy field. To this end, major protein spots were not different between paddy field and upland field among all cultivars. It could be suggested that protein expression is not severely different by various environment, but different environment affects degree of protein expression.

• Microbiology and Biotechnology Letters
• /
• v.37 no.2
• /
• pp.105-109
• /
• 2009

Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.

• Microbiology and Biotechnology Letters
• /
• v.41 no.1
• /
• pp.17-25
• /
• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, $2{\times}(Gly_4Ser_1)$ linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.67 no.4
• /
• pp.211-221
• /
• 2022

Recently, the demand for the cultivation of upland soil has been increasing, and the rate of conversion of paddy soil into upland soil is also increasing. Theincrease in uneven precipitation due to climate change has resulted in dramatic effects of waterlogging stress on upland crops. Therefore, the present study was conducted to investigate the changes in growth characteristics and the expression patterns of proteins at the two-leaf stage of adzuki beans. The domestic cultivar, Arari (Miryang No. 8), was used to test waterlogging stress. At the two-leaf stage of adzuki beans, plant height slightly decreased androot fresh weight showed significant changes after 3 days of waterlogging treatment. Chlorophyll content was also significantly different after 3 days of waterlogging treatment compared to its content in control plants. Using two-dimensional gel electrophoresis, more than 400 protein spots were identified. Twenty-one differentially expressed proteins from the two-leaf stage were analyzed using linear trap quadrupole-Fourier transform-ion cyclotron resonance mass spectrometry. Of these 21 proteins, 9 were up-regulated and 12 were down-regulated under waterlogging treatment. Protein information resource (https://pir.georgetown.edu/) categories were assigned to all 49 proteins according to their molecular function, cellular component localization, and biological processes. Most of the proteins were found to be involved in the biological process, carbohydrate metabolism and were localized in chloroplasts.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.60 no.1
• /
• pp.114-122
• /
• 2015

Soybean is very useful crop to supply vegetable protein for human. However, cultivation arear of this economically important crop is gradually diminished in upland field. Hence, cultivation area of soybean is increased in paddy field. During the growth duration of soybean, excessive moisture injury is serious problem for sustainable production and supply. We investigated protein expression according to different period of seed soaking and germination after seed soaking. For comparison on expression of protein according to different condition, we performed two-dimensional electrophoresis. After electrophoresis analysis, we selected differentially expressed protein spots according to different condition such as soaking period and germination after soaking to identify protein function by using MALDI-TOF. Results revealed that pattern of expression of protein according to soaking period and germination after soaking were generally not different in major spots. However, degree of expression of protein in some protein spots was increased in accordance with decrease of soaking period. Especially, in Hwangkeum-Kong, Danyeop-Kon, and Pecking, the degree of expression of protein was remarkably increased for 4 days after soaking. But, according to germination after soaking, degree of expression of protein in germinated seeds of all cultivars was higher than un-germinated seeds. In results of MALDI-TOF analysis, specific proteins were identified by different soaking period such as Allergen Gly m Bd 28K, P24 oleosin isoform B. Also, in accordance with germination, degree of protein expression of the related protein, Gibberellin was increased in un-germinated seeds of Iksan-Kong. In ungerminated seeds of Sinpaldal-kong, proteins were identified as down-regulated by soaking such as ATP binding and Inhibitor II', proteinase.

• Journal of Periodontal and Implant Science
• /
• v.30 no.4
• /
• pp.737-746
• /
• 2000

본 연구는 hemin이 치주질환 주요 병인균주 중의 하나인 Prevotella intermedia의 성장 및 세포막 단백질의 발현에 미치는 영향을 규명하고, hemin 결합에 관여하는 것으로 추정되는 단백질의 순수분리 및 특성 분석을 위해 수행 되었다. 본 연구의 결과에 의하면, hemin은 P.intermedia의 성장 및 세포막 단백질의 발현에 영향을 미쳐, hemin이 고갈된 조건에서 균주의 성장이 현저히 억제되었으며, 약 50 kDa의 세포막 단백질이 현저히 강화되어 발현되었다. 본 연구에서는 hemin 결합에 관여하는 것으로 추정되는 이 50 kDa의 세포막 단백질을 순수분리하였으며, N'-terminal 아미노산 분석을 수행한 결과 이 단백질은 Streptococcus inter - medius의 Enolase와 아미노산 서열 및 분자량이 일치하였다. 한편, 이 단백질에는 disulfidebond가 존재하지 않았다. 본 연구는 P.intermedia에서의 porphyrin 생리 및 hemin 획득기전을 밝히는데 있어 중요한 의의가 있으리라 사료된다.

• The Journal of Korean Society of Virology
• /
• v.26 no.2
• /
• pp.141-150
• /
• 1996

본 연구에서는 $CD4^+$ T 세포를 일회성으로 감염시킬 수 있고 $CD4^+$ T 세포에 HIV-1 envelope 유전자를 전달할 수 있는 바이러스 입자를 생산하는 HIV-1 complementation system을 개발하였고 이 system을 이용하여 $CD4^+$ T 세포에 선택적으로 HIV-1 envelope 당단백질을 발현시켰다. 이 system은 Gag/Gag-Pol expressor와 Env expressor로 구성되어있다. Gag/Gag-Pol expressor는 바이러스 입자 생산에 필요한 구조단백질과 기능단백질을 발현시키지만 packaging signal이 결핍되어 바이러스 입자로 유전자가 들어가지 못하도록 제조되었다. Env expressor는 Tat, Rev와 envelope 당단백질을 발현시키고 packaging signal을 갖고 있어 바이러스 입자로 envelope 유전자가 들어가도록 제조되었다. Gag/Gag-Pol expressor로부터 Gag와 Gag-Pol의 발현은 Rev 단백질을 요구하였고 Env expressor로부터 Rev 단백질 이 제공될 때 Gag와 Gag-Pol 단백질은 효율적으로 발현되었다. Gag/Gag-Pol과 Env expressor로 cotransfection된 COS-1 세포에서 $CD4^+$ T 세포를 일회성으로 감염시킬 수 있는 바이러스 입자가 생산되었다. 생산된 바이러스 입자는 $CD4^+$ T 세포에 HIV-1 envelope 유전자를 전달하여 envelope 당단백질을 발현시켰고 복제 가능한 자손 바이러스의 생성을 유도하지 못하였다. 본 연구에서 개발된 방법은 $CD4^+$ T 세포에서 envelope 당단백질의 기능을 분석하고 관심 있는 유전자를 $CD4^+$ T 세포에 전달하는 바이러스 입자의 생산에 이용할 수 있다.

• KSBB Journal
• /
• v.21 no.2
• /
• pp.118-122
• /
• 2006

In this report, we describe that the use of GroEL/GroES-enriched S30 extract remarkably enhances the solubility and enzymatic activity of cell-free synthesized rPA, which requires the correct formation of 9 disulfide bonds for its biological activity. We found that the stable maintenance of redox potential is necessary, but not sufficient for the optimal expression of active rPA. In a control reaction without using additional molecular chaperones, most of the rPA molecules were aggregated almost instantly after their expression and thus failed to exhibit the enzymatic activity. However, by the use of GroEL/GroES-enriched extract, combined with IAM-treatment, approximately $30{\mu}g/ml$ of active rPA was expressed in the cell-free synthesis reaction. This result not only demonstrates the efficient production of complex proteins, but also shows the control and flexibility offered by the cell-free protein synthesis system.

• The Korean Journal of Food And Nutrition
• /
• v.17 no.1
• /
• pp.47-52
• /
• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• The Journal of the Korean dental association
• /
• v.37 no.7 s.362
• /
• pp.517-529
• /
• 1999

본 연구는 태령 19일된 백서 태자 두 개관에서 분리한 골모세포유사세포에 화학발암물질인 7,12-Dimethylbenz(a)anthracene (DMBA: 0.5 ㎍/ml) 및 tumor promotor인 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 1.0 ㎍/ml)를 단독 혹은 복합 처리하여 PTRCC-DMBA, RCC-DMBA 및 RCC-DMBA-TPA 세포주를 확립시키고, 각 세포의 세포형태, 세포성장곡선, alkaline phosphatase와 acid phosphatase 활성 및 in vitro tumorigenicity를 연구하였다. 또한 c-myc, c-랜, c-jun, p53 및 Rb 유전자의 발현변화와 항암단백질인 p53 및 pRb 단백질의 발현변화를 관찰하여 골모세포유사세포가 악성형질전환되는 분자기전의 일단을 연구하고자 시행하였다. 본 실험에 사용한 모든 세포군에서 높은 aikaline phosphatase 활성과 낮은 acid phosphatase/alkaline phosphatase ratio를 보여 골모세포의 특성을 나타내었다. RCC-DMBA와 RCC-DMBA-TPA 세포는 정상세포나 PTRCC-DMBA에 비해 빠른 성장속도를 보였으며, 또한 SOFT AGAR상에서 colony를 형성하여 anchorage-independent growth를 나타내었다. 화학발암 물질로 악성변형된 세포들은 정상세포나 PTRCC-DMBA 세포에 비해 c-myc 유전자의 과발현이 관찰되었다. 정상세포에서 p53 유전자의 발현은 1.9 kb의 message만이 발현되었다. 그러나 화학발암물질로 형질전환된 세포에서는 1.9 kb message외에도 1.6 kb의 message가 더 발현되었으며, message의 양도 현저히 증가되었다. p53 단백질의 발현은 RCC-DMBA-TPA 세포에서 정상세포에 비해 현저히 감소하였으나, RCC-DMBA 세포에서는 유사한 경향을 보였다. Rb 유전자의 발현은 RCC-DMBA-TPA 세포에서만 현저히 감소하였으나, Rb 단백질의 발현은 정상세포에 비해 형질전환된 세포들에서 모두 현저히 감소되었고, 특히 RCC-DMBA-TPA 세포에서는 거의 발현되지 않았다. 이상의 결과에서 백서 태자 두 개관에서 분리한 골모세포유사세포는 화학발암물질인 DMBA에 의해 악성형질전환이 유도되었으며, c-myc의 과발현 및 p53과 Rb 단백질의 발현감소가 정상 골모세포유사세포의 악성변형과정에 밀접히 연관되어 있음을 시사한다.