Poster Ⅰ : PART A : Proteins - Structure , Functions & Modifications ; Intragenic Suppressors for the Export - Defective Signal Sequence Mutation of Ribose - Binding Protein isolated as Candidates for Folding Mutations

송택선,이영희,김정호,박찬규 한국생화학분자생물학회 (구 한국생화학회) 1991 추계학술대회집 Vol.- No.-

메탄올을 이용하여 성장하는 Methylovorus sp. strain SS1이 생산하는 세포외 다당류

추원호,송택선,김영민 한국미생물학회 1997 미생물학회지 Vol.33 No.2

제한통성 메탄올 자호세균인 Methylovorus sp. strain SS1은 최적 성장 조건하에서는 소량의 세포외 다당류(EPS)를 생산하였지만, 질소원이 결핍된 성장 조건하에서는 성장속도는 느렸지만 다량의 EPS를 생산하였다. EPS는 배지내의 탄소대 질소 비율이 5.2일 때 가장 많이 생산되었다. EPS 생산을 위한 최적 온도는 30^{\circ}C.$이고 최적 pH는 6.5였다. EPS는 탄수화물과 단백질 및 약간의 피루브산으로 구성되어 있었고, 환원당으로는 다량의 포도당과 소량의 mannose가 존재하였다. 에탄올을 처리한 EPS(EPSae)에는 에탄올을 처리하지 않은 EPS(EPSbe)에 존재하던 피루브산이 존재하지 않았고, EPS보다 단백질의 양도 적고 점성도 낮았다. EPSbe의 점성은 NaCl에 의해 큰 영향을 받았는데, 0.5%(w/v) 농도의 NaCl 용액에서도 점성이 크게 떨어졌으며, 높은 온도에서는 점성이 비가역적으로 크게 증가하였다. Gel filtration 방법으로 조사한 EPSae의 분자량은 $2.5{\times}$10^6$ - $3.5{\times}$10^6$이었다. 냉동건조한 다당류를 전자현미경으로 관찰하였을 때, EPSbe는 섬유모양을 하였고, EPSae는 벌집모양의 망상구조를 하고 있었다. Mrthylov~orits sp. starin SSl, a restricted facultative methylotrophic bacterium. growing on methanol was found to produce small amount of extracellular polysaccharide (EPS) under the optimal growth conditions, while it produced large amount of the polysaccharide under nitrogen limihtion. The optimal ratio of carbon to nitrogen for EPS production were found to be 5.2. The optimal temperature and pH for EPS production were 30^{\circ}C.$ and 6.5, spectively. The EPS consisted of carbohydrate, protein and small amount pyruvic acid. The reducing sugars in the EPS consisted mainly of glucose and a small amount of mannose. The EP!; treated with ethanol (EPSae) was found to have several properties different from those of the EPS which was not treated with ethanol (EPSbe); the EPSae contained no pyruvic acid. It also contained less protein and showed much lower viscosity than the EPSbe. The viscosity of EPSbe was very sensitive to NaCl and decreased t;harply upon exposure of the polysaccharide to even 0.5% (wiv) NaCl solution. The viscosity, however, was increased irreversibly upon exposure of the saccharide to high temperature. The molecular weight of EPS was estimiited to be $2.5{\times}$10^6$ - $3.5{\times}*$10^6$ using Sepharose hB column chromatography. Scanning electron microscopy revealed that the lyophilized EPSbe and EPSae have a structure of thread-like fibers and a mesh-like structure resembling bee-hive, respectively.

Restriction Map of a Small Plasmid in Pseudomonas carboxydovorans

김영민,송택선,Kim, Young-Min,Song, Taek-Seon 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

Pseudomonas carboxydovorans에는 2.8 kb의 small plasmid (pYK 100)가 존재하는데, 이 plasmid에는 plasmid가 curing된 세균에서 유도되는 일산화탄소산화효소의 변형과 관계된 유전자가 존재하는 것으로 알려져 있다. 본 연구에서는 AluI, MboI, NruI, SstII, HinfI, NcoI, AvaI, HaeII, HincII 등의 제한효소를 사용하여 이 plasmid의 제한효소 지도를 만들었다. 제한효소 중에서 MboI과 NruI, SstII, HinfI, NcoI, AvaI, HaeII 자리는 AluI 자리를 기준으로 하여 이들 효소를 복합적으로 사용, 여러번의 절단실험을 하여 결정하였고, HincII 자리는 AluI, HincII, SstII, HinfI을 복합적으로 사용하여 얻은 pYK100 DNA 조각과 HincII로 부분절단한 pYK100 DNA 조각의 크기를 비교분석, AluI 자리를 기준으로 그 위치를 결정하였다. 그리고 P. carboxydovorans에는 세포 하나에 약 두개의 pYK100가 존재하고 있는 것으로 밝혀졌다. The small plasmid pYK100 (2.8 kb) of Pseudomonas carboxydovorans is known to carry gene(s) for modification of carbon monoxide dehydrogenase in plasmid-cured cells of the same strain. The plasmid was physically mapped with several restriction endonucleases AluI, MboI, NruI, sstII, HinfI, NcoI, AvaI, HaeII, and HincII. The cleavage sites for MboI, NruI, SstII, HinfI, NcoI, AvaI, and HaeII were determined by multiple digestion mapping of these sites with regard to the AluI site. A HindI fragment map was obtained by analysis of pYK100 digested with combinations of AluI, HincII, SstII, and HinfI in conjunction with partial digestion mapping of HincII sites in relation to the AluI site. The copy number of pYK1000 was determined to be around two per cell using pBR322 as a standard.

대장균 리보스 결합단백질의 신호배열 변이에 대한 숙성체 부위의 회복돌연변이

이영희,송택선,김정호,박순희,박찬규 한국미생물학회 1991 미생물학회지 Vol.29 No.5

A mutational alteration in the signal sequence of ribose-binding protein (RBP) of Escherichia coli, rbsB103, completely blocks the export of the protein to the periplasm. Intragenic suppressors for this mutation have been selected on minimal medium with ribose as a sole carbon source. Six suppressor mutations were characterized in detail and were found to have single amino acid wubstitution in the mature portion of RBP, which resulted in the mobility shift of the proteins on SDS polyacrylamide gel. Amino acid changes of these suppressors were localized in several peptides which are packed to form the N terminal domain of typical bilobate conformation of RBP. The involvement of SecB, a molecular chaperone, was investigated in the suppression of signal sequence mutation. Translocation efficency was found to be increased by the presence of SecB for all suppressors. It is likely that the folding characteristics of RBP altered by the suppressor mutations affect the affinity of interaction between SecB and RBP.

Carboxydobacteria 를 위한 재조합 Plasmid 백터와 형질전환방법 개발

김진욱,송택선,김영민 한국미생물학회 1992 미생물학회지 Vol.30 No.3

Carboxydobacteria 의 일산화 탄소 산화에 대한 유전학적 연구를 위해 Pseudomonas caarboxydovorans 에 존재하는 pYK100 plasmid 와 pBR322 를 이용하여 pYK322 (7.2 kb, Ap, Tc) 와 pYK324 (7.2 kb, Ap, Tc) 등 두가지 재조합 plasmid shuttle 백테를 만들고, pYK100와 pACYC184를 이용하여 pYK210(5.2 kb, $CM^{r}$ ), pYK220 (5.2kb,$CM^{r}$ ), pYK230 (5.2 kb, $Cm^{r}$ ), pYK232 (5.2 kb, $CM^{r}$) 등 네가지 shuttle 벡터를 만들었다. 재조합된 벡터들은 보두 대장균에서 안정되게 복제되었다. pYK322 와 pYK220 을 이용한 carboxydobacteria 의 형질전환 실험에서 Bagdasarian 과 Timmis 의 방법 (Curr. Top. Microbiol. Immunol., 96 :47-67, 1982) 을 변형하여 0.2% succinate 가 포함된 무기염류배지에서 지수성장 중기까지 배댜ㄷ한 세균을 이용하고, 형질전환용액의 10 mM RbCI 을 100 mM KCI 로 대체하며, 형질전환용액 처리후 4.deg.C 에서의 방치시간을 12시간으로 하고, DNA첨가휴 45.deg.C 에서 3 분간 heat shock 을 준 경우에 높은 형질전환이 일어났다. 형질전환된 세균으로 부터 형질전환에 사용한 plasmid 를 발견할 수 없었는데, 이는 도입된 plasmid 가 염색체 DNA 에 결합되었기 때문인 것으로 추측된다. Recombinant plasmid shuttle vectors were constructed for genetic studies on the oxidation of carbon monoxide by carboxydobacteria. Two vectors. pYK322 (7.2 kb, Ap'. Tc') and pYK 324 (7.2 kb, Ap', Tc'), were constructed using pBR322 and pYK100. a small plasmid in Pseudomonas carbo,xydovorans. Four plasmids. pYK2IO (5.2 kb. Cm'), pYK220 (5.2 kb, Cmr), pYK230 (5.2 kb, Cm'), and pYK232 (5.2 kb. Cm'), were constructed using pACYC184 and pYK100. Transformation of several carboxydobacteria with pYK322 and pYK220 was round to be efficient when the cells were transformed by the methoti of Bagdasarian and Timmis (Curr. Top. Microbiol. Immunol. 96:47-67. 1982) with several modifications; cells growing on 0.2% succinate were harvested at the mid-exponential phase. 10 mM RbCl in transformation solution was substituted with 100 mM KCI. cclls in transformation solution were incubated for 12 h at 4'C before addition of DNA and heat shock was carried out for 3 min at 45$^{\circ}$C. Plasmid vectors used for transformation, however. were not detected from antibiotics-resistant transformants, suggesting that the vectors may be integrated into the chromosomal DNA.

Pseudomonas carboxydovorans 에 존재하는 small plasmid의 제한효소 지도

김영민,송택선 ( Young Min Kim,Taek Seon Song ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

The small plasmid pYK100 (2.8 kb) of Pseudomonas carboxydovorans is known to carry gene(s) for modification of carbon monoxide dehydrogenase in plasmid-cured cells of the same strain. The plasmid was physically mapped with several restriction endonucleases AluI, MboI, NruI, SstII, HinfI, NcoI, AvaI, HaeII, and HincII. The cleavage sites for MboI, NruI, SstII, HinfI, NcoI, AvaI, and HaeII were determined by multiple digestion mapping of these sites with regard to the AluI site. A HincII fragment map was obtained by analysis of pYK100 digested with combinations of AluI, Hincll, SstII, and HinfI in conjunction with partial digestion mapping of HincII sites in relation to the AluI site. The copy number of pYK1000 was determined to be around two per cell using pBR322 as a standard.

단백질의 Folding 을 늦추는 아미노산 잔기의 치환이 단백질 분비에 미치는 영향

김종호,강문석,이덕연,송택선,백광희,박찬규 ( Jong Ho Kim,Moon Seog Kang,Duk Yeon Lee,Taek Sun Song,Kwang Hee Baek,Chan Kyu Park ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.2

A mutational change in the signal sequence of ribose-binding protein (RBI of E. coli blocks the export of this protein to the periplasm. Intrngenic suppressors for the mutation that have single amino acid substitution in the mature portion of RBP at Ala-27 (A27T) and Val-50 have been characterized previously. In order to assess the role of these amino acids in protein folding and translocation, site-specific mutagenesis approach was employed to substitute various amino acids in those positions. Eight amino acid changes for the position 27, and 11 for the position 50 were obtained. Chemotactic assay showed that 5 out of 8 changes at 27 and 11 of 11 changes at 50 restored taxis to ribose. Pulse-labelling and chase experiment demonstrated that, among RBPs with the suppressor mutation, less stable proteins tend to be more efficiently exported to the periplasm.

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay

제승모,Un-chang Yeo,송택선,Ki-cheol Kim,Sung-Yun Park,Man-Jung Kim,조상래 대한수의학회 2014 Journal of Veterinary Science Vol.15 No.2

The interferon-gamma (IFN-γ) assay is employed as acomplementary diagnostic test for bovine tuberculosis(BTB) in many countries. To simplify this assay, weestablished a 96-well plate format using the ESAT-6 andCFP-10 antigens and then employed it to determine theextent of Mycobacterium (M.) bovis infection in dairy herdswith a history of BTB outbreaks in a country where onlyselective culling is practiced. The sensitivity and specificityof this IFN-γ assay were 85.9% and 100%, respectively,based on comparison with the conventional singleintradermal tuberculin test (SIDT). The IFN-γ assay wasalso positive in 30.4% and 36.8% of SIDT-negative animalsfrom herds with recent and remote BTB outbreaks,respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five(35.7%) were culture positive and an additional six werepositive based on a polymerase chain reaction-based test forM. bovis. Therefore, the IFN-γ assay has the potential toserve as a specific and sensitive test for M. bovis infection indairy cattle. Further, the results indicated that a substantialportion of SIDT-negative animals in herds with previousBTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy mayrequire modifications to include this more sensitive assay.

Characterization of a Novel Antigen of Mycobacterium tuberculosis K strain and Its Use in Immunodiagnosis of Tuberculosis

Paul J. Park,김아름,Yangkyo P. Salch,송택선,신성재,한승정,조상래 한국미생물학회 2014 The journal of microbiology Vol.52 No.10

Mycobacterium tuberculosis-specific antigens would be ofgreat value in developing immunodiagnostic tests for tuberculosis(TB), but regional differences in molecular types ofthe organism may result in antigenic variation, which in turnaffects the outcome of the tests. For example, the Beijingstrains of M. tuberculosis are prevalent in East Asia, and inparticular, the K strain and related strains of the Beijingfamily, are most frequently isolated during school outbreaksof TB in South Korea. From comparison of genome sequencesbetween M. tuberculosis K strain and the H37Rv strain, anon-Beijing type, we identified a K strain-specific gene, InsB,which has substantial homology with the ESAT-6-like proteins. This study was, therefore, initiated to characterize theInsB protein for its immunogenicity in mice and to confirmits expression in TB patients by detecting antibodies to theprotein. The InsB gene was cloned from M. tuberculosis Kstrain and expressed in Escherichia coli. The recombinantInsB protein was used for immunization of mice. All miceshowed strong antibody responses to the InsB protein, andsplenocytes stimulated with InsB showed strong IFN-γ andIL-17 responses and a weak IL-2 response, all of which havebeen implicated in disease expression and used for the immunodiagnosisof TB. Serum samples from TB patients alsoshowed significant antibody responses to the InsB protein ascompared to healthy control samples. These results indicatethat the InsB protein is an M. tuberculosis K-strain-specificantigen that could further improve the current immunodiagnosticmethods, especially for the South Korean population.