유전자조작, 균주분리 대장균을 이용한 Akt/PKB Protein Kinase의 발현 및 활성화

이재학 ( Jae Hag Lee ) 한국미생물생명공학회 2009 한국미생물·생명공학회지 Vol.37 No.2

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℃ 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.

한천 분해효소를 생산하는 해양 미생물 SH-1의 분리 및 특성 분석

이재학 ( Jae Hag Lee ),이순열 ( Soon Youl Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2014 한국미생물·생명공학회지 Vol.42 No.4

한천을 분해하는 해양미생물을 한천을 유일한 탄소원으로 하는 인공 해수 한천 배지를 이용하여 제부도 개펄에서 분리하였다. SH-1으로 명명한 분리된 균주는 그람음성균이며 한 개의 극성 편모를 가지는 균이었다. 16S rRNA 유전자의염기서열의 유사성 분석 결과 분리된 균주는 Neiella marina J221 [9]과 가장 높은 상동성을 보였다(96.5%). 분리 균주는 28-37oC에서 생장하였지만 42oC에서는 생장하지 못하였고 한천분해효소의 활성은 37oC보다 28oC에서 높은 활성을 보였다. 또한 SH-1균주는 1-5% NaCl (w/v)를 포함하는 배양액에서 생장이 가능했으며 3%의 농도에서 가장 좋은 생장을 보였고 한천을 분해하는 효소의 활성도 3% 염분농도의 배양액에서 가장 높았다. 48시간 배양한 세포배양액을 농축하여 조효소액을 준비하여 효소의 적정 pH와 적정 온도를 조사한 결과 pH 7.0에서와 40oC에서 최적 효소 활성을 보였다. 조효소액을 사용하여 zymogram 분석을 실시한 결과 분자량 15, 35, 52 KD 크기의 3개 이상의 한천 분해효소를 생산하는 것으로 보인다. 박막크로마토그라피(TLC) 분석 결과 아가로스를 분해하여 네오올리고당을 생성하는 β-agarase를 생산하는 것으로 추정된다. A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-negative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at 28-37ºC but not at 42ºC, and the agarase activity of the cell culture supernatant was higher when grown at 28ºC than when grown at 37ºC. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agardegrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and temperature for agar-degrading activity at 7.0 and 40ºC, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces β-agarase to degrade agarose to neoagarooligosaccharides.

스페인어 동사변이형 -ria와 -ra에 대한 고찰

이재학 ( Lee Jae Hag ) 한국스페인어문학회 2003 스페인어문학 Vol.0 No.29

A trave´s de la historia de la lengua espan~ola las variantnes verbale -ri´a y -ra se han alternado en ciertos contextos y muchos grama´ticos han cuestionado este feno´meno. Esta alternancia se debe a la confunsio´n de la funcio´n gramatical entre la -ri´a y la -ra. Histo´ricamente se han vacilado estas dos variantes verbales entre el valor indicativo y el subjuntivo en ciertos contextos y han salido numerosos estudios para definir su categori´a gramatical. En los siglos anteriores la Real Academia Espan~ola ha definido la -ri´a como subjuntivo al apoyar la idea de Antonio Nebrija. Pero, despue´s de que Andre´s Bello ha postulado que la -ri´a no pertenece al subjuntivo sino al indicativo, muchos grama´ticos como Gili y Gaya han apoyado su idea. Al comienzo del siglo XX la misma RAE tambie´n ha abandonado su postulacio´n de los siglos anteriores y ha anunciado que la -ri´a pertenece a un nuevo modo llamado potencial. Asi´ es como el debate sobre la funcio´n gramatical de esta variate verbal tan curiosa nunca parece terminar. Sea cua´l sea su definicio´n grama´tical, la -ri´a comparte ciertos rasgos con la -ra y esto no es debe al contacto lingu¨i´stico sino al valor modal que consiguio´ en la apo´dosis de la cla´usula condicional en el lati´n vulgar. Pues, el presente trabajo analiza los estudios anteriores sobre la -ri´a y examina su alternancia con la -ra. Al mismo tiempo trata de probar que las dos variantes han conseguido el valor modal a trave´s del desarrollo etimolo´gico de la lengua espan~ola. Finalmente este trabajo postula que las -ri´a y -ra se pueden alternar libremente en los contextos en que se extesa la probalidad en las situaciones irreales y la futuridad en el timpo pasado.

고려인삼의 마이크로파 처리 효과

이재학 ( Jae Hag Lee ),금준석 ( Jun Seok Kum ) 한국식품영양학회 2010 韓國食品營養學會誌 Vol.23 No.3

The effect of microwave treatment on Korean ginseng was studied by measuring the changes in moisture, crude lipid, crude ash, crude protein, total dietary fiber and saponin contents, as well as changes in density, color and microstructure. Korean ginseng was treated with 100 or 200 watts of microwaves for 1 or 3 hrs, respectively, followed by drying using an oven at 60℃ for 96 hrs. The moisture contents decreased to 13.12~10.77% from an initial 76.26%. The amounts of lipid and ash were reduced in proportion to the time of microwave treatment and level of microwave power. The amount of protein in ginseng after microwave treatment did not significantly change. The amount of total dietary fiber increased after microwave treatment and the color of dried ginseng became dark. The amounts ofginsenoside-Rb1, Rb2+Rb3, Rc, Rd, Re, Rf, Rg1, Rg2+Rh1 and Rg3 were reduced after treatment with 100 watts of microwave radiation for 1 and 3. The amounts ofginsenoside-Rb1, Rd, Re, Rf, Rg1, Rg2+Rh1 and Rg3 after treatment with 200 watts of microwave radiation for 1 and 3 hr also reduced. On the other hand, the amounts ofginsenoside-Rb2+Rb3 and Rc after treatment of ginseng with 200 watts of microwave radiation for 1 and 3 hrs were increased.

급식소 조리종사자의 장갑 위생을 위한 표준관리기준의 연구

이재학 ( Jae-hag Lee ),배현주 ( Hyun-joo Bae ),이재경 ( Jae-kyoung Lee ) 한국고등직업교육학회 2005 한국고등직업교육학회논문집 Vol.6 No.2

Most of elementary, middle and high school have had getting a contract with one of foodservice industries. The more foodservice industries expanded, the more foodborne diseases occurred. A major risk factor for the diseases is gloves that are used by cook. The purpose of this study was to study the standard operation for the glove hygiene of cook in cafeteria. Gloves of the cook in two cafeterias occupied one elementary and one middle school, were tested. The samples from the gloves were prepared by Glove-Juice method. Gloves were tested for microbiological contamination before and in the middle of cook. They were contaminated with Coliform bacterias as well as general bacterias. Standard operation manual of the disinfection method for glove hygiene was made and instructed to cook and performed as it is. The gloves used by the cook were tested again after hygiene instruction to cook and disinfection of the gloves by standard operation. Hygiene of the gloves before cook was much improved and some of the gloves had no contamination of Coliform bacteria. However, hygiene of gloves in the middle of cook was not much improved. In conclusion, hygiene instruction for cook and standard operation for disinfection of the gloves is necessary for the prevention of foodborne diseases in the cafeterias. It is recommended that gloves used in the middle of cook should be changed, periodically.

Mammaglobin 유전자 재조합 및 발현에 관한 연구

이재학 ( Jae Hag Lee ) 한국식품영양학회 2004 韓國食品營養學會誌 Vol.17 No.1

Glutathione S-Transferase에 융합한 재조합 Hybrid Peptide Gaegurin-LL37의 대장균에서의 발현

바야르바트이쉬반질 ( Bayarbat,Ishvaanjil ),이재학 ( Jae Hag Lee ),이순열 ( Soon Youl Lee ) 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 2012 한국미생물·생명공학회지 Vol.40 No.2

항균 펩타이드(Antimicrobial peptides(AMPs)는 그람 양성, 그람 음성 세균과 진균병원체에 대항하는 생명체에서 중요한 역할을 하는 물질이다. 인간의 Cathelicidin 항균 펩타이드는 임상학적으로 사용할 수 있는 여러 가지의 생물학적 활성을 가진다. 항균 펩타이드의 생산 비용은 재조합 방법으로 낮출 수가 있다. 대장균은 저렴하며 손쉬운 조작이 가능하기 때문에 다른 재조합 단백질처럼 항균 펩타이드의 발현에 훌륭한 숙주가 될 수 있다. 그러나 대장균에서의 항균 펩타이드의 과발현은 항균 펩타이드가 과발현 되었을 때 대장균에 독성을 보일 수 있으므로 어려움이 보고가 되어있다. 본 연구에서는 이러한 문제점을 극복하고자 항균 펩타이드를 Glutathione S-transferase(GST) 결합 단백질에 융합하여 항균펩타이드의 독성을 감소시키도록 설계하여 발현을 시도하였다. 이 때 발현한 항균 펩티드는 LL37과, gaegurin4과 LL37의 잡종 펩타이드 GGN4-LL37(GL32로 명명)를 GST에 융합되도록 벡터를 구축하고 설계하여 대장균에서 GST 융합단백질로 발현시켰다. 융합 단백질은 친화력 컬럼을 사용하여 분리하고 GST를 절단하여 항균펩타이드 만을 분리하였고 분리한 펩타이드는 웨스턴 블롯팅으로 확인하였고 그람 양성, 그람 음성 세균에 대하여 항균 활성을 나타내는 것을 확인하였다. Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.

수조사육에 의한 청충, Perinereis aibuhitensis (Grube)의 산란과 난발생

강경호(Kyoung Ho Kang),이재학(Jae Hag Lee),장영진(Young Jin Chang),유성규(Sung Kyoo Yoo) 한국수산과학회 양식분과 1997 韓國養殖學會誌 Vol.10 No.1

종양 대치물을 이용한 사지 구제술의 방사선학적 평가

문상호,이상훈,김한수,오주한,고병원,구기형,이재학,황정수,이한구,Moon, Sang-Ho,Lee, Sang-Hoon,Kim, Han-Soo,Oh, Joo-Han,Ko, Byung-Won,Koo, Ki-Hyung,Lee, Jae-Hag,Hwang, Chung-Soo,Lee, Han-Koo 대한근골격종양학회 2000 대한골관절종양학회지 Vol.6 No.4

목적 : 악성 골종양의 종양 대치물을 이용한 사지 구제술에서 방사선학적 변화를 평가하는 데 목적이 있다. 대상 및 방법 : 종양 대치물로써 사지 구제술을 받은 69례를 대상으로 하였고 평균 추시 기간은 4년 9개월이었다. International Symposium On Limb Salvage(ISOLS)에서 제창한 방사선학적 대치물 평가 체계(Radiological Implants Evaluation System)를 사용하여 평가하였으며 술후 1년 및 최종 추시로 나누어 시행하였다. 우수, 양호, 보통, 불량을 각각 통계분석을 위해 4, 3, 2, 1로 변환하여 분석하였고 평가 결과를 시멘트 사용 여부, 병변의 위치, 종양의 종류, 종양 대치물의 종류 및 나이(<20세 vs ${\geq}$20세)에 따라 나누어 T-test 및 ANOVA를 사용하여 비교 분석하였다. 결과 : 최종 추시에서 시멘트군이 무시멘트군보다 골 재형성 및 접촉면 항목에서 우수했으나 고정 항목(anchorage)에서는 무시멘트군이 우수했다. 병변의 위치 상 근위 대퇴부 및 원위 경골부가 다른 부위보다 골 재형성면에서 불리하였으며 골육종군이 비골육종군보다 고정 및 관절면에서 불리하였다. 대치물의 종류 및 연령에 따른 유의한 차이는 없었다. 결론 : 방사선학적 중기 추시상, 시멘트를 이용한 재건술이 골 흡수 및 해리에서 더 우수하였고 무시멘트 술식은 고정(anchorage) 면에서 우수하였다. 원위 대퇴골 및 근위 경골이 골 흡수면에서 유리한 해부학적 위치였으며 골육종군은 화학요법으로 인해 고정 및 관절면에서 불리하였다고 생각한다. Purpose : To verify radiological changes of limb salvage operation using tumor prosthesis. Materials and Methods : Sixty-nine cases which used tumor prosthesis were reviewed. They have been followed up for average four years and nine months. We evaluated radiographs by ISOLS(International Symposium On Limb Salvage) radiological implants evaluation system immediate postoperatively, at postoperative 1 year and last follow-up. After converting 'excellent, good, fair, poor' to '4, 3, 2, 1' for stastical analysis, data were analyzed according to the use of cement, anatomical sites, diagnosis implant designs and age (<20 year vs. ${\geq}$20 year) using t-test and ANOVA. Results : The outcomes of cemented type prostheses were superior in remodelling and interface but inferior in anchorage. The scores of proximal femur and distal tibia were worse in bone remodelling. Osteosarcoma group had lower scores in anchorage and implant articular problem. There were no significant differences according to four different implant designs and age. Conclusion : In the intermediate term follow-up radiological evaluation, cemented type prosthesis were better in the aspects of bony resorption and osteolysis, and cementless one in anchorage. Distal femur and proximal tibia have higher scores in bony resorption, and osteosarcoma was worse tumor entity in anchorage and articular problem, and chemotherapy may be the cause.

전문대학 2002학년 신입생의 실태조사 분석

강현중 ( Hyun-joong Kang ),이원근 ( Won-geun Lee ),이재학 ( Jae-hag Lee ) 한국고등직업교육학회 2003 한국고등직업교육학회논문집 Vol.4 No.2

We implemented the survey for actual conditions of College Freshmen. Among total 3553 freshmen in S college, 3081(86.7%) student replied to the questionnaire for the Characteristics of the new student. Total 38 survey items in 4 survey areas including individual characteristic, life, self-concept, course decision. We presented percentage by day classes, night classes and industry group classes and described its meanings. Finally, we abridged results of this surrey by 4 survey areas.