• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• 한국발생생물학회지:발생과생식
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• 제10권3호
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• pp.177-184
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• 2006

레티놀 결합 단백질(retinol-binding protein, RBP)은 고등 척추동물에서 혈류를 통해 특이적으로 레티놀을 표적세포에 운반해 주는 중요한 역할을 한다. 우리나라의 연안에 서식하고 있으며 산업적으로 중요한 조피볼락(Sebastes schlegeli)을 대상으로 4-nonylphenol(NP)가 RBP mRNA 발현에 미치는 영향을 구명하기 위해 간으로부터 cDNA library를 제작하고 RBP 단편의 염기서열을 분석하였다. 분석된 RBP mRNA 서열로부터 아미노산 서열을 추정하여 다른 종의 RBP 아미노산 서열과 비교한 결과 Sparus aurata와는 80%, 무지개 송어(Oncorhynchus mykiss)와는 72%, 유럽 뱀장어(Anguilla anguilla)와는 78%의 높은 상동성을 보였다. 조피볼락에서 4-NP에 대한 RBP와 VTG mRNAs 발현에 미치는 영향을 northern blot 분석 방법에 의해 조사하였다. 4-NP를 10 mg/kg BW로 주사한 실험구에서는 암수 모두 VTG mRNA 발현에 영향을 주지 않았으나, RBP mRNA 발현은 수컷에서 48시간 후에 감소하였다. 4-NP를 25 mg/kg BW으로 주사한 실험구에서는 24시간 후에 암수 모두 VTG mRNA 발현이 증가하였으며, RBP mRNA 발현은 48시간 후에 암수 모두 감소하였다. 이들 결과로부터 조피볼락에서는 4-NP 등의 에스트로겐 유사물질이 RBP와 VTG 유전자 발현에 상반되는 효과를 유도함을 알 수 있었다.

• 한국식품영양학회지
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• 제9권4호
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• pp.452-458
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• 1996

호열균 B. stearothermophilus의 세포내 PPIase를 정제하여 Edman 법으로 N-말단 아미노산 배열을 결정하여 이를 바탕으로 합성한 올리고누클레오티드의 프리머를 이용하여, 서턴 분석하여 PPIase 유전자 약 3.0kb를 클로닝하였다.(pPI-40) PPI-40으로부터 PPIase N-말단 배열을 코드 하는 영역으로부터 합성한 프리머(A-1, B-2)를 이용하여, PCR법으로 PPIase N-말단을 코드 하는 유전자를 증폭하여, 염기배열을 경정한 후, 그 정보에 따라 유전 해석한 결과 PCR로 증폭된 단편(pSN-18)은 165염기로부터 형성된 55 아미노산잔기를 코드 하는 open reading frame (ORF)이 계속되고 있었고, Edman법으로 결정한 PPIase N-말단 아미노산 39 아미노산잔기가 완전히 일치하였다. 그리고, 이 ORF를 중심으로, 지금까지 클론화된 대장균의 PPIasea (cytoplasm)와 PPIase b(periplasm)의 아미노산 일차구조 해석으로부터 각각 58%(cytoplasm), 16%(periplasm)의 상동성을 나타냈다. PPIase 구조 유전자를 갖는 재조합플라스미드 pPI-40을 JM109로 형질전환하여 Lac 프로모터로 PPIase 단백질을 발현시켰다. 효소 분자량을 SDSPAGE로 확인한 결과 약 18kDa으로 호열균 B. stearothermophilus로부터 정제한 단백질 분자량과 동일하다. 면역억제(CsA, FK506)와의 화학적인 반응은 대장균의 PPIase와 동일하게, 면역억제와는 비감수성으로 나타났다.

• IMMUNE NETWORK
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• 제3권3호
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Plant Biotechnology Reports
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• 제4권2호
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• pp.165-172
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• 2010

Genes that are expressed early in specific response to high salinity conditions were isolated from rapeseed plant (Brassica napus L.) using an mRNA differential display method. Five PCR fragments (DD1.5) were isolated that were induced by, but showed different response kinetics to, 200 mM NaCl. Nucleotide sequence analysis and homology search revealed that the deduced amino sequences of three of the five cDNA fragments showed considerable similarity to those of ${\beta}$-mannosidase (DD1), tomato Pti-6 proteins (DD5), and the tobacco harpin-induced protein hin1 (DD4), respectively. In contrast, the remaining clones, DD3 and DD2, did not correspond to any substantial existing annotation. Using the DD3 fragment as a probe, we isolated a full-length cDNA clone from the cDNA library, which we termed BnSWD1 (Brassica napus salt responsive WD40 1). The predicted amino-acid sequence of BnSWD1 contains eight WD40 repeats and is conserved in all eukaryotes. Notably, the BnSWD1 gene is expressed at high levels under salt-stress conditions. Furthermore, we found that BnSWD1 was upregulated after treatment with abscisic acid, salicylic acid, and methyl jasmonate. Our study suggests that BnSWD1, which is a novel WD40 repeat-containing protein, has a function in salt-stress responses in plants, possibly via abscisic acid-dependent and/or -independent signaling pathways.

• BMB Reports
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• 제34권3호
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• pp.262-267
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• 2001

A cDNA coding alkaline phosphatase (AP) homologue was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The nucleotide sequence of the cloned cDNA appeared to lack the N-terminal coding region. The genomic DNA encoding alkaline phosphatase homologue was isolated from S. pombe chromosomal DNA using PCR. The amplified DNA fragment was ligated into plasmid pRS315 to generate the recombinant plasmid pSW20. The DNA insert was subcloned as two smaller fragments for nucleotide sequencing. The sequence contains 2,789 by and encodes a protein of 532 amino acids with a molecular mass of 58,666 daltons. The S. pombe cells containing plasmid pSW20 showed much higher AP activity compared with the yeast cells with vector only This indicates that the cloned AP gene apparently encodes AP The predicted amino acid sequence of the S. pombe AP shares homology with those of other known APs.

• 식물조직배양학회지
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• 제28권5호
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• pp.283-287
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• 2001

감자 (Solanum tuberosum L. cv. Irish Cobbler)의 괴경형성과정 (tuberization) 동안에 발현하는 유전자들의 발현양상을 조사하고자 differential display법을 실시하였다. Differential display를 이용하여 분리된 eIF5A DNA단편을 probe로 사용하여 감자의 cDNA library screening을 통하여 eIF5A full-length cDNA를 감자에서 처음으로 분리하였다. 감자의 eIF5A, clone은 토마토의 eIF5A cDNA 염기서열과 94.8%. 아미노산 서열에서는 97.5%로 매우 높은 유사성을 나타내었다. 감자의 eIF5A 유전자는 길이가 716 bp로 하나의 단백질 code영역 (ORF)을 포함하고 있었다. 이 영역은 분자량 17.4 kD, pI 5.5로 추정되는 160개의 아미노산으로 구성된 eIF5A단백질을 code하고 있었다. eIF5A 단백질들에서 12개의 아미노산 서열 (STSKTGKHGHAK)은 효모에서 사람에 이르기까지 완벽하게 보존되어 있는 것으로 알려져 있는데, 감자에서도 또한 잘 보존되어 있었다. 이 영역은 eIF5A 단백질의 활성을 나타내는 데 있어서 필수적인 hypusine을 생성하는 전사 후 수식 부위가 들어 있는 아주 중요한 곳이다. 감자에서 eIF5A 유전자의 발현양상을 조사한 결과 감자의 전조직에서 발현을 보였는데, 성숙잎이나 괴경보다는 세포분열 및 물질축적이 활발히 일어나고 있는 꽃기관들 (stamen, ovary, petal. sepal), 과실 (fruit)과 stolen 등의 조직들에서 비교적 활발히 발현되고 있었다.

• Animal cells and systems
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• 제3권2호
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• pp.229-236
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• 1999

The recA gene plays a central role in genetic recombination and SOS DNA repair in Escherichia coli (E. coli). We have previously identified a 42 kDa RecA-like protein inducible by a variety of DNA damages from a fluorescent Pseudomonas strain sp. and characterized its inducible kinetics. In the present study, we cloned and characterized the gene encoding the RecA-like protein by immunological screening of Pseudomonas genomic expression library using polyclonal E. coli anti-RecA antibodies as a probe. From 10$^{5}$ plaques screened, five putative clones were finally isolated. Southern blot analysis indicated that four clones had the same DNA inserts and the recA-like gene was located within the 3.2 kb EcoRI fragment of Pseudomonas chromosomal DNA. In addition, the cloned recA-like gene was transcribed into an RNA transcript approximately 1.1 kb in size, as judged by Northern blot analysis. The cellular level of RNA transcript of the cloned recA-like gene was increased to an average of 5.15- fold upon treatment with DNA damaging agents such as ultraviolet (UV)- light, nalidixic acid (NA), methyl methanesulfonate (MMS), and mitomycin-C (MMC). These results suggest that the cloned gene is inducible by DNA damage similarly to the recA gene in E. coli. However, the cloned gene did not restore the DNA damage sensitivity of the E. coli recA-mutant.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.153-159
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• 2001

In order to clone the peptide synthetase gene form Lysobacter lactamgenus IFO 14,288, the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-$\alpha$-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L. lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis formt he obtained pPTS-5 cosmid showed th presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (YbjZ) and the metal ion uptake protein of Bacillus subtilis (YvrN). A 45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis. 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the hose cell growth, probably due to the disturbance of the membrane transport system.

• Journal of Microbiology
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• 제44권1호
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.