인체 ${\alpha}1$-antitrypsin cDNA의 분리 및 E.coli 내에서의 발현

이상철,유명희,Lee, Sang-Chul,Yu, Myeong-Hee 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

인체 간 cDNA library로부터 올리고 뉴클레오티드 프로브를 이용하여 ${\alpha}1$-antitrypsin (${\alpha}1$-AT) cDNA를 지닌 파지 클론을 스크린하였다. 선별된 클론에는 1.3 k bp의 DNA가 삽입되어 있었으며 이를 pUC9 벡터에 다시 클론닝하여 분석한 결과 이미 보고된 ${\alpha}1$-AT cDNA 서열과 일치함을 알 수 있었다. ${\alpha}1$-AT cDNA를 E. coli에서 발현시키기 위해 택 (tac) 프로모터에 의해 전사가 활성화되도록 발현벡터 pT-AT를 제조하였다. 플라스미드 pT-AT에 의해 형질전환된 E. coli JM109 균주에서 재조합 단백질의 발현을 유도한 결과 ${\alpha}1$-AT의 활성이 검증되었으며, 발현된 ${\alpha}1$-AT는 사람 피에서 정제한 ${\alpha}1$-AT와 그 면역학적 특성이 동일함을 알 수 있었다. 또한 같은 세포 추출액을 SDS-polyacrylamide 젤 전기영동법으로 분석한 결과 Coomassie Brilliant Blue로 단백질 밴드를 염색했을 때는 발현된 ${\alpha}1$-AT를 검증할 수 없었으나, ${\alpha}1$-AT에 대한 rabbit IgG 항체와 펄옥시다제가 연결된 anti-rabbit IgG goat 항체를 사용하여 웨스턴 블릿을 한 결과 ${\alpha}1$-AT 밴드가 기대했던 분자량 위치에서 확인되었다. 이와 같이 생산된 재조합 ${\alpha}1$-AT는 본래의 ${\alpha}1$-AT보다 분자량이 작은 것으로 나타났는데 이는 E. coli 세포내에서 발현된 ${\alpha}1$-AT는 glycosylation이 안된 형태의 것이기 때문일 것이다. Human ${\alpha}1$-AT cDNA was screened from human liver cDNA library, by use of oligonucleotide probes. DNA insert of 1.3 kbp from a positive clone was subcloned into pUC9 plasmid and was analyzed by restriction enzyme digestion and partial DNA sequencing. The obtained gene was identical to human ${\alpha}1$-AT cDNA reported previously. In order to express ${\alpha}1$-AT cDNA under regulation of tac promoter, expression vector pT-AT was constructed. E. coli JM109 cells transformed with pT-AT showed ${\alpha}1$-AT activity upon induction of recombinant protein synthesis. The recombinant ${\alpha}1$-AT was immunologically identical to authentic ${\alpha}1$-AT that was purified from human blood. When the recombinant cell lysates were analyzed on SDS-polyacrylamide gel electrophoresis, the expressed ${\alpha}1$-AT was not detected by staining with Coomassie Brilliant Blue. However Western blotting of the same gel, using rabbit IgG against ${\alpha}1$-AT and peroxidase-conjugated goat anti-rabbit IgG antibody, showed the existence of ${\alpha}1$-AT at the expected molecular weight region. Molecular weight of recombinant ${\alpha}1$-AT was slightly lower than that of authentic ${\alpha}1$-AT; which presumably was due to lack of glycosylation inside E. coli cells.

인체 α 1 - antitrypsin cDNA 의 분리 및 E . Coli 내에서의 발현

이상철,유명희 ( Sang Chul Lee,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Human α1-AT cDNA was screened from human liver cDNA library, by use of oligonucleotide probes. DNA insert of 1.3 kbp from a positive clone was subcloned into pUC9 plasmid and was analyzed by restriction enzyme digestion and partial DNA sequencing. The obtained gene was identical to human αl-AT cDNA reported previously. In order to express αl-AT cDNA under regulation of tac promoter, expression vector pT-AT was constructed. E. coli JM109 cells transformed with pT-AT showed αl-AT activity upon induction of recombinant protein synthesis. The recombinant αl-AT was immunologically identical to authentic αl-AT that was purified from human blood. When the recombinant cell lysates were analyzed on SDS-polyacrylamide gel electrophoresis, the expressed α1-AT was not detected by staining with Coomassie Brilliant Blue. However Western blotting of the same gel, using rabbit IgG against αl-AT and peroxidase-conjugated goat anti-rabbit IgG antibody, showed the existence of αl-AT at the expected molecular weight region. Molecular weight of recombinant αl-AT was slightly lower than that of authentic αl-AT; which presumably was due to lack of glycosylation inside E. coli cells.

재조합 베타갈락토시다제 - 프리 S2 융합단백질에 존재하는 두 개의 메치오닌 위치에 올리고뉴클레오티드를 이용한 변이유도

박종광,이상철,최영철,한문희,유명희 ( Chong Kwnag Park,Sang Chul Lee,Young Chul Choi,Moon H . Han,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1

E. coli JM109 cells harboring pCTHB20 plasmid overproduced β-galactosidase-preS2 (βgal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation of preS2 peptide out of the fusion protein after CNBr cleavage, the lacZ sequence was rearranged to construct pCTHB30 plasmid. Two internal methionine codons in the lacZ region of pCTHB20 and pCTHB30 was also substituted with valine codons by oligonucleotide-directed mutagenesis. The EcoRI-XbaI restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mp18 vector. Two synthetic complementary oligonucleotides (17-mer each) with desired nucleotide changes were used simultaneously as primers in synthesizing a complementary strand. Mutant clones were screened by DNA hybridization with probes of ^(32)P-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coli JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced βgal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.

대장균에서 융합단백질의 대량생산을 위한 베타갈락토시다제 폴리펩티드 길이의 최적화

최영철,이상철,한문희,유명희 ( Young Chul Choi,Sang Chul Lee,Moon H . Han,Myeong Hee Y ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.1

Plasmid vectors for cloning and directing the synthesis of large amounts of fusion proteins in E. coli were constructed. These plasmids carried tac promoter, the coding sequence of amino terminal portion of β-galactosidase, and the polylinker region at the 3`-end of truncated lacZ. When E. coli MC1061 cells were transformed with these plasmids, 25-60% of the total cellular proteins were represented by the lacZ polypeptides. The overproduced proteins formed inclusion bodies inside the cells. When the amounts of recombinant proteins produced by these plasmids were examined, the higher yield was observed with those of shorter lacZ polypeptides. The length of recombinant polypeptide with the maximum yield (60%) was about 280 amino acids long. However, when the length of β-galactosidase decreased to 200 residues as in pCT30 vector, the yield of recombinant proteins decreased dramatically. When the length of polypeptide increased again to 260 residues by fusing pre-S2 sequence of hepatitis B virus to lacZ sequence in pCT30, the yield of fusion polypeptides increased to over 50% of total cellular proteins.

하시모트 갑상선염과 동반된 갑상선 MALT 림프종 ( MALToma ) 1 예

이태영(Tae Young Lee),류은상(Eun Sang Ryoo),남일송(Il Song Nam),홍기영(Gi Young Hong),한찬희(Chan Hee Han),윤석기(Suk Gi Yoon),김철희(Chul Hee Kim),변동원(Dong Won Byun),김영선(Young Sun Kim),서교일(Gyo Il Seo),유명희(Myung Hi Yoo),진 대한내과학회 2001 대한내과학회지 Vol.61 No.3

Primary thyroid lymphomas constitute of up to 5% of all thyroid malignancies. Recently, mucosa-associated lymphoid tissue (MALT) lymphoma are relatively recognized as a B cell subset of non-Hodgkin's lymphoma. MALT-lymphomas are thought to develop from acquired lymphocytic tissue during the course of a chronic inflammatory or autoimmune process. In the thyroid , which is normally devoid of lymphocytic tissue, chronic autoimmune thyroiditis (Hashimoto's disease) has been associated with an increased risk of lymphoma, including MALT type. The clinical presentations include the enlarging of the neck mass, dysphagia, hoarsenes and choking or cold thyroid nodule. We report a case of MALToma of the thyroid accompanied by Hashimoto's thyroiditis with a review of the literature.(Korean J Med 61:281-285, 2001)

Oligonucleotide-directed Mutagenesis of Two Methionine Residues in Recombinant ${\beta}$-galactosidase-preS2 Fusion Proteins

박종광,이상철,최영철,한문희,유명희,Park, Chong-Kwang,Lee, Sang-Chul,Choi, Young-Chul,Han, Moon-H.,Yu, Myeong-Hee 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

플라스미드 pCTHB20에 의해 형질전환된 대장균 JM109 균주는 베타갈락토시다제-프리 S2 융합 단백질을 총 세포 단백질의 50% 정도로 생산하였다 (Choi et al., 1988). 이 융합 단백질로부터 시안브롬을 사용하여 프리 S2 펩티드를 절단하여 정제하는 것을 보다 더 용이하게 하기 위해 베타갈락토시다제 유전자 (lacZ) 서열을 재배열하여 pCTHB30을 제조하였으며, 또한 lacZ 서열 내부에 존재하는 메치오닌 코돈을 올리고뉴클레오티드에 의한 변이유도 방법을 사용하여 발린코돈으로 치환하였다. 플라스미드 pCTHB2에 존재하는 lacZ 서열을 포함하는 EcoRI-XbaI DNA 절편을 분리하여 파지 M13 mp18 백터의 멀티크로닝 부위에 삽입하였고, 원하는 변이를 포함하는 두개의 상보쇄 서열 (각각 17-mer)을 합성하여 동시에 프라이머로 사용하였다. 변이클론들은 $^{32}P$로 표지된 변이 올리고뉴클레오티드를 프로브로 사용하여 DNA 하이브라디제이션 방법에 의해 선별하였으며, 치환된 뉴클레오티드는 DNA 염기서열 분석에 의해 확인하였다. 변이 lacZ 서열로 치환하여 제조된 pCMHB20 및 PCMHB3에 의해 형질전환된 대장균 JM109 균주들도 pCTHB20 및 PCTHB30을 포함하는 균주와 거의 같은 효율로 프리 S2 서열을 지닌 융합 단백질을 생산하였다. E. coli JM109 cells harboring pCTHB20 plasmid overproduced ${\beta}$-galactosidase-preS2 ($\beta$ gal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation of preS2 peptide out of the fusion protein after CNBr cleavage, the lacZ sequence was rearranged to construct pCTHB30 plasmid. Two internal methionine codons in the lacZ region of pCTHB20 and pCTHB30 was also substituted with valine codons by oligonucleotide-directed mutagenesis. The EcoRI-XbaI restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mp18 vector. Two synthetic complementary oligonucleotides (17-mer each) with desired nucleotide changes were used simultaneously as primers in synthesizing a complementary strand. Mutant clones were screened by DNA hybridization with probes of $^{32}P$-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coli JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced ${\beta}$ gal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.

Optimization of the ${\beta}$-galactosidase Polypeptide Length for the Overproduction of Fusion Proteins in E. coli.

최영철,이상철,한문희,유명희,Choi, Young-Chul,Lee, Sang-Chul,Han, Moon-H.,Yu, Myeong-Hee 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

대장균 내에서 융합단백질을 다량 생산하기 위한 발현벡터들이 개발되었다. 이들 플라스미드 벡터들은 tac 전사활성화 서열, 베타갈락토시다제의 아미노 말단을 코우드하는 뉴클레오티드 서열 그리고 그의 3'말단에 클로닝 부위로서 폴리링커를 지니고 있다. 대장균 MC1061 균주를 이들 플라스마드로 형질전환 시키고 재조합 단백질의 생산을 유도하였을 때 절단된 베타갈락토시다제 폴리펩티드의 양은 전체 세포단백질의 25-60% 정도가 되었다. 이렇게 생산된 재조합 단백질들은 세포내에서 함유체 (inclusion body) 상태로 축적되었다. 이들 플라스미드에 의해 생산되는 폴리펩티드의 수율을 조사하였을 때 길이가 짧을수록 수율이 올라감을 알 수 있었는데, 길이가 280개 아미노산 정도일 때 최대의 수율(60%)을 나타냈다. 하지만 pCT30 플라스미드에서와 같이 길이가 200개 아미노산 정도로 너무 짧아지면, 수율이 현저하게 감소하였다. 이 pCT30 플라스미드의 베타갈락토시다제 서열의 3'말단에 간염 바이러스의 pre-S2 서열을 연결시켜, 생산되는 융합 폴리펩티드 길이를 260개 아미노산으로 늘어나게 하였을 때 융합 단백질의 수율은 다시 총 세포 단백질의 50% 이상으로 증가하였다. Plasmid vectors for cloning and directing the synthesis of large amounts of fusion proteins in E. coli were constructed. These plasmids carried tac promoter, the coding sequence of amino terminal portion of ${\beta}$-galactosidase, and the polylinker region at the 3'-end of truncated lacZ. When E. coli MC1061 cells were transformed with these plasmids, 25-60% of the total cellular proteins were represented by the lacZ polypeptides. The overproduced proteins formed inclusion bodies inside the cells. When the amounts of recombinant proteins produced by these plasmids were examined, the higher yield was observed with those of shorter lacZ polypeptides. The length of recombinant polypeptide with the maximum yield (60%) was about 280 amino acids long. However, when the length of ${\beta}$-galactosidase decreased to 200 residues as in pCT30 vector, the yield of recombinant proteins decreased dramatically. When the length of polypeptide increased again to 260 residues by fusing pre-S2 sequence of hepatitis B virus to lacZ sequence in pCT30, the yield of fusion polypeptides increased to over 50% of total cellular proteins.

대장균내에서 발현된 가용성 IL-6의 정제 및 Alanine-Scanning Mutagenesis에 의한 IL-6 Mutant의 제조

이상철,유명희,이민주,변광호,최인표 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.4

Interleukin-6(IL-6) is a multifunctional cytokine that acts on various target cells to induce a diversity of biological responses. Studies were performed on the purification of soluble and functional IL-6 expressed in the periplasm of Escherichia coli. The coding region of the IL-6 gene was fused to the pe1B secretion signal sequence and the expression was under the control of T7 promotor. Upon cell induction under 26 C, recombinant IL-6 was overexpressed in the periplasmic soluble fraction. The periplasmic soluble extract was already 50% pure and was further purified to homogeneity by gel filtration and Mono-Q column chromatography. About 1.5 mg pure protein was obtained from 11 cultured cells. The recombinant IL-6 had a specific activity_ of 1 x 108 U/mg in B9 bioassay, which is eqivalent to that of the natural IL-6. To understand the structure-functional relationship of IL-6, twenty two alanine substitutions of IL-6 have been generated in helix B and surrounding loop regions (amino acid residues 51-109). Most of the mutant proteins were soluble, but those having alanine substitution at Asp7l, G1u93, Phe 94, G1u95, Tyr97, and Arg104 showed little expression or the reduced yield, suggesting that these residues may involve in the folding process or the stability of IL-6 tertiary structure.

포상기태(Hydatidiform mole)에 의해 발생된 심한 갑상선중독증 1예

이재학,박종근,권순효,목지오,윤지성,김여주,박형규,김철희,김상진,이해혁,남계현,권계현,고은석,변동원,서교일,유명희 대한내분비학회 2003 Endocrinology and metabolism Vol.18 No.4

저자들은 무월경의 11주의 27세 환자에서 심한 갑상선중독증을 동반하고, β-HCG가 1,123,0001U/L으로 크게 증가되었으나, 흡입소파술 시행후 임상 증상과 갑상선기능 그리고 β-HCG가 모두 정상으로 호전된 포상기태 (Hydatidiform mole) 1예를 경험하였기에 문헌 고찰과 함께 보고하는 바이다. Human chorionic gonadotropin(HCG) is one of the glycoproteins families synthesized by the placenta, and consists of 2 noncovalently joined subunits, namely, α' and . The α' and -subunits have a structural homology with the α' and -subunits of TSH and LH. The thyrotropic action of HCG results from its structural similarity to TSH, so -HCG can bind to the TSH receptor in the thyroid gland. A high level of HCG, accompanied by an increased thyroid hormone level, can be observed in gestational trophoblastic diseases (GTD), such as a hydatidiform mole or a chorio- carcinoma. However, the clinical symptoms of hyperthyroidism in GTD are rarely observed. A 27-years-old woman, admitted due to an amenorrhea of 11 weeks duration, with thyrotoxic symptoms, such as weight loss, palpitation, sweating, tremor, heat intolerance and anxiety, was evaluated. Her serum free T4 level was 8 times higher than normal, and her serum -HCG level was over l,OO0,00OIU/L. She had a curettage operation, with the pathological findings of a complete hydatidiform mole. These thyrotoxic symptoms developed due to a hydatidiform mole, and were accompanied with a highly increased serum β-HCG level. After evacuation of the molar tissue, the thyroid hormone and thyrotoxic symptoms normalized. Here, this case is reported, with brief review of the literature (J Kor SOC Endocrinol 18:420425, 2003).