• BMB Reports
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• 제32권3호
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• pp.306-310
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• 1999

The partial cDNA of the MT-c clone encoding snake venom metalloprotease was subcloned and expressed in E. coli. The expressed metalloprotease was purified by affinity chromatography in the presence of urea, and then successfully refolded into its functional form, retaining metalloprotease activity that hydrolyzes fibrinogen. The simple and convenient refolding strategy established in this work was highly efficient in recovering the recombinant enzyme activity. Experimental evidence suggests that the C-terminal amino acid stretch of 16 residues is a critical sequence for proper folding of the metalloprotease domain.

• Archives of Pharmacal Research
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• 제15권1호
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• pp.48-51
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• 1992

Achromobacter xylosoxidans KF701 and Pseudomonas putida (NAH7) were significantly different in degradative capability of aromatic compounds including benzoates, biphenyls, and naphthalene. However, both of the bacterial strains can grown on catechol as the sole carbon and energy source. Catechol 2, 3-dioxygenase gene for naphthalene oxidation or biphenyl oxidation was cloned into Escherichia coli HB 701. A E. coli HB 101 clone containing catechol 2, 3-dioxygenase gene from P. putida (NAH7) contains a recombinant plasmid with 3.60kb pBR322 and 6-kb insert DNA. Another E. coli HB101 clone containing catechol 2, 3-dioxygenase gene from A. xylosoxidans KF 701 has a recombinant plasmid with 4.4kb pBR322 and 10-kb insert DNA. Physical maps of the recombinant plasmids were constructed, and catechol 2, 3-dioxygenase gene in the recombinant plasmide was further localized and subcloned int M13. The cloned-catechol 2, 3-dioxygenase game products were identified as yellow bands on nondenaturaing polyacrylamide gel after electrophoresis followed by activity staining with catechol solution.

• Journal of Microbiology and Biotechnology
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• 제17권6호
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• pp.879-890
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• 2007

The identification of tumor antigens is essential for the development of anticancer therapeutic vaccines and clinical diagnosis of cancer. SEREX (serological analysis of recombinant cDNA expression libraries) has been used to identify such tumor antigens by screening sera of patients with cDNA expression libraries. SEREX-defined antigens provide markers for the diagnosis of cancers. Potential diagnostic values of these SEREX-defined antigens have been evaluated. SEREX is also a powerful method for the development of anticancer therapeutics. The development of anticancer vaccines requires that tumor antigens can elicit antigen-specific antibodies or T lymphocytes. More than 2,000 antigens have been discovered by SEFEX. Peptides derived from some of these antigens have been evaluated in clinical trials. This review provides information on the application of SEREX for identification of tumor-associated antigens (TAA) for the development of cancer diagnostics and anticancer therapeutics.

• 한국미생물·생명공학회지
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• 제27권4호
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• pp.278-285
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• 1999

In order to use heat shock promoter for the detection of toxic substances, dnaK promoter was amplified from E. coli genomic DNA by using a polymerase chain reaction(PCR) followed by sequencing and sub-cloning into the multi-cloning site of the plasmid, pUCD615. The pUCD615 is a broad-host-range vector containing promoterless lux operon originated from V.fischeri. The recombinant plasmid was transfered to E. coli DH5$\alpha$ through electroporation. The recombinant E. coli showed several patterns of bioluminescent responses to ethanol stress. The bioluminescent E. coli also showed responses to other toxic substances including FeK3(CN)6, CdCl2, p-nitrophenol and HgCl2. The increases of RLU(Relative Light Unit) were observed at 100ppm of FeK3(CN)6, 10ppm and 100ppm and 100ppm of CdCl2, 1ppm of 10ppm of p-nitrophenol and at 1ppm of HgCl2.

• 한국연초학회지
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• 제23권2호
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• pp.133-140
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• 2001

PAP-I cDNA was synthesized from total RNA of Phytolacca americana leaves by RT-PCR, and then subcloned to recombinant vector pBluescript II SK-. Using PCR with primers designed in our laboratory, we could get the 9 deletion mutant PAP-I cDNA fragments. The first of the fragments was deleted by 66bp from immature N-terminal and then the rest were deleted by 90bp sequentially. Sequentially deletion mutant PAP-I cDNAs were inserted to pAc55M, on down-stream of gall promoter. Recombinant pAc55M was transformed to yeast cells, psy1 and the cells were spreaded on SC_urn-/glucose plate media. Colonies on SC_ura-/glucose plate were streaked on the same position of SC_ura-/glucose and SC_ura-/galactose plate, and we selected colonies growing on both plates, which carry non-cytotoxic deleted mutant PAP-I cDNA. We selected 4 deletion mutant PAP-I cDNAs which have not cytotoxicity.

• 한국미생물·생명공학회지
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• 제22권1호
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• pp.18-22
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• 1994

Enterococcus faecalis KBL703 has three plasmids(p703/9, p703/5 and p703/4). Within p703/5, the specific DNA region that would confer replication function(replication origin) was searched by transformation experiments. In order to use as the recipient of transformation, two plasmid-cured strainsd were made from this strain. Four recombinant DNA constructs, each containing fragment of p703/5 and CAT(chloramphenicol acetyl transferase) gene were also made. And they were used to transform the plasmid-cured strains. Only one DNA construct containing 3.6 kb SalI fragment was stably maintained as plasmid in these strains. Additional experiment using another Enterococcus faecalis strain(ATCC29212) as a recipient was successfully done and it was confirmed that this newly constructed recombinant plasmid plasimid contained the replication origin from p703/5 plamid.

• 미생물학회지
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• 제25권3호
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• pp.165-172
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• 1987

An expression vector in a mammalian cell was constructed using the origin of replication (OR) and the promoters of SV40. The plasmid pSVOE was constructed by inserting SV40 DNA fragment (1, 118bp) containing SV40 OR and promoters into pBR322-1, and then a multiple cloning sequence was inserted at the immediate downstream of the late promoter of SV40 in the pSVOE vector. The plasmid was named pSVML. As a selection marker, thymidine kinase gene of herpes simplex virus with its promoter was inserted into EcoRI site of pSVML and the recombinant was named pSVML-TKp. To test the expression capacity of foreigen gene inserted at the multiple cloning site of pSVML, the thymidine kinase gene without its own promoter was inserted at the BamHI site of pSVML. The recombinant was named pSVML-TK. These plasmids, pSVML-TKp and pSVML-TK, were transfected into COS cells with calcium phosphate precipitation method. The thymidine kinase activity was significantly increased in both transfected cells.

• 생명과학회지
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• 제14권2호
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• pp.362-367
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• 2004

장염비브리오균의 숙주 내 감염을 일으키는 기작을 이해하기 위하여 세포외 효소 중의 하나인 콜라겐분해효소의 유전자가 불활성화된 돌연변이체를 제작하였다. 콜라겐분해효소의 유전자인 vppC 유전자에 항생제 내성 유전자인 nptII를 삽입하여 제작된 재조합 DNA를 suicide vector인 pDMS197에 클로닝하여 pVCM03이라 명명하였다. 재조합 suicide 플라스미드 pVCM03을 E. coli 7213에 형질전환하여 접합을 통하여 원 균주인 V. varahaemolyticus 04에 전달하였다. 전달된 pVCM03 유래의 재조합 vvpC::npfII DNA는 homologous recombination에 의해 wild-type allele와 교환되어 돌연변이체를 형성하게 되고, 돌연변이체는 10% sucrose가 함유된 TCBS 배지에서 선별되었다. Allele exchange는 PCR에 의한 증폭된 DNA의 크기 비교로 확인하였다. 돌연변이체인 V. parahaemolyticus CM은 원 균주와 비교하였을 때 약 4배정도 낮은 콜라겐 분해 활성을 나타내었고, vero cell을 이용한 MTT assay에서도 원 균주에 비하여 낮은 세포독성을 보였다.

• 한국동물학회지
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• 제39권3호
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• pp.248-256
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• 1996

본 연구의 목적은 인간의 Poly(ADP-ribose) synthetase (PARS)의 cDNA를 클로닝하여 발현시키기 위해 수행하였다. 먼저, 인간의 PARS cDNA 전체를 포함한 pPARS3.1을 pGEM-7Zf(+) 등의 발현 벡터 클로닝하였다. 이 결과로 생성된 재조합 플라스미드 pPARS6.1이 인간의 PARS cDNA 전체를 포함하고, 올바른 방향으로 삽입되었는지를 확인하기 위해 제한효소 지도를 작성하였고, random primed DNA probe을 이용한 Southern blot 분석에 의해서 PARS가 클로닝되었다는 것을 확인하였다. 또한, 염기서열 분석 결과, 단백질 합성이 시작되는 유전 암호가 정확한 순서로 위치하고 있음을 확인하였다. 재조합된 pPARS6.1의 발현을 위해 배양시 0.4 mM IPTG로 처리하여 만들어진 인간의 PARS 단백질이 10% SDS-PAGE에서 120 kDa 위치에 이동하였다는 것을 nick-translated된 DNA를 probe로 이용하여 확인하였고, Southwestern blot 실험 결과 120 kDa과 98kDa에 위치하는 단백질이 DNA와 결합함을 알 수 있었다.

• 대한수의학회지
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• 제39권4호
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• pp.797-805
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• 1999

T sergenti cDNA library were constructed to get a more broad information about the structural, functional or antigenic properties of the proteins, and analyzes for their partial cDNA sequences and expression sequences tags(ESTg). The mRNA were purified from T sergenti isolates to identify the information of antigen gene, then first and second strand cDNA was synthesized. EcoR I adaptor ligation and Xho I enzyme restriction were used to the synthesized cDNA, and ligated into a Uni-ZAP XR vector. T sergenti cDNA library was constructed with packaging and amplification in vitro. Antibody screening was performed with constructed T sergenti cDNA library using antisera against T sergenti. Among those clones, eight phagemids were rescued from the recombinant in vivo excision with f1 helper phage. Using the analysis of endonuclease restriction and PCR, the recombinant cDNA were proved having a 0.5-3.0kb of inserts. The eight of partial cDNA clones' sequences were obtained and examined for their homology using BLASTN and BLASTX. The eight of sequenced clones were classified into three groups according to the basis of database searches. A total 3,045bp of partial cDNA sequence were determined from six clones. The putatively identified clones contain a cytochrome c gene, a heat shock protein gene, a cyclophilin gene, and a ribosomal protein gene. The unidentified clones have a homology to ATP-binding protein(mtrA) gene of S argillaceus, DNA-binding protein(DBP) gene of Pseudorabies virus 85kDa merozoite protein gene of B bovis, mRNA spm1 protein of T annulata and glycine-rich RNA-binding protein mRNA of O sativa etc.