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B형 간염 바이러스 프리 - S2 펩티드의 분리 정제 및 중합 인혈청 알부민 수용체 검출법
윤혜영,한문희,함경수 ( Hye Young Yun,Moon H . Han,Kyung Soo Hahm ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
Recombinant pre-S2 peptide was purified to apparent homogeneity from E. coli by immunoaffinity chromatography using monoclonal antibody and studied for its binding characteristics with monoclonal antibody and pHSA (polymerized human serum albumin). A simple and sensitive assay method for detecting the activity of pHSA receptor on pre-S2 peptide of hepatitis B virus surface antigen (HBsAg) has been developed using pre-S2-β-galactosidase fusion protein. The assay can be used for determining pre-S2 or factors neutralizing pHSA receptor activity. The sensitivity of the assay is about 10 to 100 pmol of pre-S2.
Recombinant Human Interleukin-2: II. Biological and Therapeutic Activities
최혜림,윤혜영,함경수,Choi, Hye-Lim,Yun, Hye-Young,Talmadge, James E.,Hahm, Kyung-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2
Recombinant human interleukin-2 (rH IL-2, $Ser^{125}$-rH IL-2) purified from E. coli was characterized its biological and therapeutic activities for the establishment of preclinical screening system and therapeutic applications. In vitro augmentation of natural killer (NK) cell activity and mixed lymphocyte reaction (MLR) assay were shown to be comparable with other rH IL-2 from different manufacturers and laboratories. An antiserum against rH IL-2 was obtained from chick, and characterized the reacticvity with rH IL-2 reconstituted from lyophilized state in relation to the stability and conformational changes of the recombinant protein. In addition, long term storage of rH IL-2 in lyophilized or solution was not shown to incur remarkable loss of MLR activities, and any appearance of either degradation or aggregation products when analyzed by SDS-PAGE, indicating the stability and suitability for therapeutic applications of rH IL-2. 대장균으로 부터 분리정제된 재조합 인체 인터루킨-2 (KAIST rH IL-2, $Ser^{125}$-rH IL-2)를 전임상시험 및 암치료에 응용하기 위한 목적으로 생물학적 및 면역요법적 활성도를 측정하였다. In vitro에서 자연살해세포 활성의 증진능력 및 혼합 임파구반응 실험에서 활성을 나타냈으며 다른 회사의 재조합 인터루킨-2 제품들과 유사한 결과를 얻었다. 재조합 인체 인터루킨-2의 입체구조적 및 생물학적 활성도의 안정성을 조사하기 위하여 정제된 재조합 인체 인터루킨-2에 대한 항혈청을 닭에서 얻었으며 동결건조 저장하였던 재조합 인체 인터루킨-2와 이 항체와의 반응을 조사하였으며, 또한 6개월간 저장하였던 인터루킨-2의 안정성을 혼합 임파구반응 및 전기영동 방법으로 그 활성 및 단백질 성분을 조사한바 혼합임파구 반응에서 여전히 활성을 보여주였으며, 전기영동상 분해되었거나 응집된 산물이 생기지 않았음을 확인하였다. 이상의 결과는 KAIST 재조합 인체 인터루킨-2가 전임상시험 및 임상시험 목적에 사용하기에 적합함을 시사한다.
윤혜영,한문희,함경수,Yun, Hye-Young,Han, Moon-H.,Hahm, Kyung-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
대장균내에서 과량 발현된 재조합 프리-S2 펩티드를 면역친화력 크로마토그래피에 의해 분리 정제하였으며, 이 펩티드에 대한 중합 인혈청 알부민(pHSA)의 결합을 연구하였다. 프리-S2 부위의 pHSA수용체 활성을 검출하는 방법을 프리-S2-$\beta$ 갈락토시다제 융합단백질을 이용하여 개발하였으며, 민감도는 10-100 p mol의 프리-S2에 해당되었다. 또한 이 방법은 프리-S2 농도 결정 뿐 아니라 pHSA수용체 활성을 중화시킬 수 있는 인자들을 측정하는데 이용될 수 있다. Recombinant pre-S2 peptide was purified to apparent homogeneity from E. coli by immunoaffinity chromatography using monoclonal antibody and studied for its binding characteristics with monoclonal antibody and pHSA (polymerized human serum albumin). A simple and sensitive assay method for detecting the activity of pHSA receptor on pre-S2 peptide of hepatitis B virus surface antigen (HBsAg) has been developed using pre-S2-$\beta$-galactosidase fusion protein. The assay can be used for determining pre-S2 or factors neutralizing pHSA receptor activity. The sensitivity of the assay is about 10 to 100 pmol of pre-S2.
B 형 간염바이러스 표면항원 중 Pre - S2 부위의 항원구조 및 중합 알부민 수용체의 특성
윤혜영,박필련,김승목,함경수 ( Hye Young Yun,Pil Yon Park,Seung Moak Kim,Kyung Soo Hahm ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.2
Pre-S2 region (amino acids 120-174) of hepatitis B virus surface antigen contains immunodominant and disulfide bond-independent epitopes. Using pre-S2 peptides (recombinant peptides 121-174/adw2, 134-170/adw2 and synthetic peptide 120-145/ayw), epitope and polyalbumin receptor region on pre-S2 were studied. Polyalbumin receptor assay and enzyme immunoassay provided evidences that the region of at least 12 residues (134-145) of pre-S2 was shared by epitope for an anti-pre-S2 monoclonal antibody and polyalbumin receptor. This region was shown to be hydrophilic and a possible β-sheet.
윤혜영,박필련,김승목,함경수,Yun, Hye-Young,Park, Pil-Yon,Kim, Seung-Moak,Hahm, Kyung-Soo 생화학분자생물학회 1990 한국생화학회지 Vol.23 No.2
B형 간염바이러스(HBV) 표면항원 단백질(HBsAg) 중 55개의 아미노산으로 구성된 pre-S2 부위를 재조합 대장균으로부터 순수 분리하였다. 이 부위의 펩티드 절편들(121-174/adw2, 134-170/adw2, 120-145/ayw)은 anti-pre-S2(adw2) 단일클론항체 및 중합 인혈청 알부민에 결합하는 결과를 보였으며 이는 세 펩티드의 공통서열인 134-145 부위에 항원 결정부위 및 중합 인혈청 알부민 수용체가 존재하는 것을 암시한다. 134-145 부위는 HBV의 여러 serotype들에서 보존된 서열을 포함하며, 2차구조로 예측 결과 ${\beta}$-sheet 형성의 높은 가능성을 보인다. 또한 anti-pre-S2 단일클론항체가 pre-S2의 중합 인혈청 알부민 수용체 활성을 저해시키는 결과는 HBV 중화항체에 대한 항원 결정부위와 중합 인혈청 알부민 수용체 부위가 같거나 공통서열을 가짐을 뒷받침한다. Pre-S2 region (amino acids 120-174) of hepatitis B virus surface antigen contains immunodominant and disulfide bond-independent epitopes. Using pre-S2 peptides (recombinant peptides 121-174/adw2, 134-170/adw2 and synthetic peptide 120-145/ayw), epitope and polyalbumin receptor region on pre-S2 were studied. Polyalbumin receptor assay and enzyme immunoassay provided evidences that the region of at least 12 residues (134-145) of pre-S2 was shared by epitope for an anti-pre-S2 monoclonal antibody and polyalbumin receptor. This region was shown to be hydrophilic and a possible ${\beta}$-sheet.
특정 위치 변이방법에 의한 Ser - interleukin - 2 - 의 생산
강성만,곽주원,권종범,김성완,이인영,이선복,윤혜영,함경수,한문희,나도선 ( Seong Man Kang,Ju Won Kwak,Jong Bum Kwon,Sung Wan Kim,In Young Lee,Sun Bok Lee,Hye Young Yun,Kyung Soo Hahm,Moon H . Han,Doe Sun Na ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.3
Human Interleukin-2 (IL-2) posessing a serine in place of cystein at the amino acid sequence position 125 has been produced in E. coli by site directed mutagenesis. The activity of purified Ser^(125)-IL-2 was more than 2.5 times higher than that of Cys^(125)-IL-2 as determined by the growth promoting effect on IL-2 dependent cell line. The yield of production of Ser^(125)-IL-2 was at least 1.5 times higher as compared to that of Cys^(125)-IL-2.
Production of $Ser^{125}$-Interleukin-2 by Site Directed Mutagenesis
강성만,곽주원,권종범,김성완,이인영,이선복,윤혜영,함경수,한문희,나도선,Kang, Seong-Man,Kwak, Ju-Won,Kwon, Jong-Bum,Kim, Sung-Wan,Lee, In-Young,Lee, Sun-Bok,Yun, Hye-Young,Hahm, Kyung-Soo,Han, Moon-H.,Na, Doe-Sun 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.3
아미노산 서열 125번 위치의 아미노산인 시스테인을 세린으로 치환한 Inter1eukin-2를 특정 위치 변이방법에 의하여 대장균을 이용하여 생산하였다. $Ser^{125}$-IL-2는 IL-2 의존성인 배양세포에 대하여 $Cys^{125}$-IL-2와 유사한 성장촉진효과를 나타내었다. $Ser^{125}$-IL-2의 생산 수율은 $Cys^{125}$-IL-2에 비하여 1.5배 이상 높았다. Human Interleukin-2 (IL-2) posessing a serine in place of cystein at the amino acid sequence position 125 has been produced in E. coli by site directed mutagenesis. The activity of purified $Ser^{125}$-IL-2 was more than 2.5 times higher than that of $Cys^{125}$-IL-2 as determined by the growth promoting effect on IL-2 dependent cell line. The yield of production of $Ser^{125}$-IL-2 was at least 1.5 times higher as compared to that of $Cys^{125}$-IL-2.
재조합 인체 인터루킨 - 2 1 . 분리정제 및 생화학적 특성
윤혜영,최혜림,이명규,김승호,나도선,이선복,한문희,함경수 ( Hye - Young Yun,Hye - Lim Choi,Myeong Kyu Lee,Seung Ho Kim,Doe Sun Na,Sun Bok Lee,Moon H . Han,Kyung - Soo Hahm ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2
Recombinant human interleukin-2 (rH IL-2, Ser^(125)-rH IL-2) was purified to apparent homogeneity from E. coli in high yield and characterized its biochemical properties for the establishment of preclinical screening system and therapeutic applications. The purification was carried out by methods involving isolation of inclusion body, urea extraction, solubilization and gel filtration chromatography. The renaturation of the product was achieved by extensive dialysis against the storage buffer. The purity was confirmed by SDS-PAGE and HPLC. Amino terminal amino acid analysis and partial amino acid sequence analysis showed that the primary structure of the recombinant protein (rH IL-2) was found to be identical with natural IL-2. The purity and the conformation of the rH IL-2 were also confirmed by obtaining crystals of the recombinant protein.
재조합 인체 인터루킨 - II . 생물학적 및 면역요법적 활성도
최혜림,윤혜영,James E . Talmadge,함경수 ( Hye Lim Choi,Hye Young Yun,James E . Talmadge,Kyung Soo Hahm ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2
Recombinant human interleukin-2 (rH IL-2, Ser^(125)-rH IL-2) purified from E. coli was characterized its biological and therapeutic activities for the establishment of preclinical screening system and therapeutic applications. In vitro augmentation of natural killer (NK) cell activity and mixed lymphocyte reaction (MLR) assay were shown to be comparable with other rH IL-2 from different manufacturers and laboratories. An antiserum against rH IL-2 was obtained from chick, and characterized the reacticvity with rH IL-2 reconstituted from lyophilized state in relation to the stability and conformational changes of the recombinant protein. In addition, long term storage of rH IL-2 in lyophilized or solution was not shown to incur remarkable loss of MLR activities, and any appearance of either degradation or aggregation products when analyzed by SDS-PAGE, indicating the stability and suitability for therapeutic applications of rH IL-2.
Recombinant Human Interleukin-2: I. Purification and Biochemical Characterization
윤혜영,최혜림,이명규,김승호,나도선,이선복,한문희,함경수,Yun, Hye-Young,Choi, Hye-Lim,Lee, Myeong-Kyu,Kim, Seung-Ho,Na, Doe-Sun,Lee, Sun-Bok,Han, Moon-H.,Hahm, Kyung-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2
유전자 재조합 방법에 의해서 대장균으로 부터 재조합 인체 인터루킨-2 (rH IL-2, $Ser^{125}$-rH IL-2)를 과발현 시켰으며, 이 재조합 인체 인터루킨-2를 순수분리하여 그 생화학적 특성을 조사하였다. 분리 및 정제는 세포봉입체의 분리, 우레아 추출, 용해 및 젤여과 크로마토그라피 등을 사용하였으며, 정제된 인터루킨-2의 원형재현은 투석법을 사용하였다. 정제된 재조합 인터루킨-2의 순도는 전기영동 및 HPLC로 확인하였으며, 또한 결정을 얻음으로써 재확인하였다. 아미노말단 아미노산분석 및 아미노산배열순서를 일부 결정함으로서 정제된 재조합 인체 인터루킨-2가 천연 인터루킨-2와 동일한 일차구조를 갖고 있음을 확인하였다. Recombinant human interleukin-2 (rH IL-2, $Ser^{125}$-rH IL-2) was purified to apparent homogeneity from E. coli in high yield and characterized its biochemical properties for the establishment of preclinical screening system and therapeutic applications. The purification was carried out by methods involving isolation of inclusion body, urea extraction, solubilization and gel filtration chromatography. The renaturation of the product was achieved by extensive dialysis against the storage buffer. The purity was confirmed by SDS-PAGE and HPLC. Amino terminal amino acid analysis and partial amino acid sequence analysis showed that the primary structure of the recombinant protein (rH IL-2) was found to be identical with natural IL-2. The purity and the conformation of the rH IL-2 were al so confirmed by obtaining crystals of the recombinant protein.