• 한국가축번식학회지
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• 제17권1호
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• pp.43-48
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• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.

• Journal of Life Science
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• 제11권2호
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• pp.94-98
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• 2001

Type I signal peptidase cleaves the signal sequence from the amino terminus of membrane and secreted proteins afters these protein insert across the membrane. This enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. Despite considerable research, the signal peptidase assay still remains improvement to provide further understanding of the mechanism and high-throughput inhibitor screening of this enzyme. In this paper, three known signal peptidase assays are tested with an E. coli D276A mutant signal peptidase to distinguish the sensitivity of each assays. In vitro assay using the procoat synthesized by in vitro transcription translation shows that the D276A signal peptidase I was inactive while in vivo processing of pro-OmpA expressed in the temperature-sensitive E. coli strain IT41 as well as in vitro assay using pro-OmpA nuclease A substrate show that D276A signal peptidase I has activity like wild-type signal peptidase. These results suggest that in vitro assay using the pro-OmpA nuclease A and in vivo pro-OmpA processing assay are more sensitive monitors than in vitro assay using the pro-coat. In conculsion, caution should be used when interpreting the in vitro results using the procoat.

• BMB Reports
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• 제34권2호
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• pp.118-122
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• 2001

The translocation of ribose-binding protein (RBP) into the inverted membrane vesicles (IMV) of Escherichia coli and eukaryotic microsomes was studied using the in vitro translation/translocation system. It was found that RBP was translocated into heterologous eukaryotic microsomes co-translationally, as well as post-translationally However, RBP was translocated only past-translationally into IMV. Degradation fragments of RBP with the molar mass of 14 and 16 kDa were produced during the translocation into IMV However, the amount of the degradation products decreased and the mature form of RBP appeared in the presence of phenylmethylsulfonyl fluoride (PMSF). PMSF and GTP accelerated the translocation of RBF It was also found that SecB enhanced the post-translational translocation of RBP It appears that RBP is translocated via at least two targeting paths.

• 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.

• Applied Biological Chemistry
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• 제32권3호
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• pp.222-231
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• 1989

대두에 존재하는 단백질 분해 효소 저해제로는 Kunitz trypsin inhibitor (SKTI), Bowman-Birk proteinase inhibitor, 그리고 그의 isoinhibitor들이 알려져 있다. SKTI는 분자량이 20K이며 181개의 아미노산으로 구성되어 있다. SKTI의 분자 구조 및 발현 특이성을 밝히기 위하여, 콩에서 순수 분리한 SKTI를 항원으로 사용하여 항체를 제조하였다. 이 항체는 항원으로 사용한 SKTI에 대하여 특이적으로 반응할 뿐만 아니라 기내에서 합성된 그의 전구체와도 특이적으로 반응하였다. 기내에서의 단백질 합성은 미성숙 대두 종자에서 mRNA를 분리한 후, wheat germ extract를 이용하여 실시하였다. 합성된 단백질 중에서 대두에 존재하는 SKTI는 검출되지 않는 반면에 면역침전법에 의해 분자량이 24K인 전구체가 존재하고 있음을 확인하였다. 이와 같이 SKTI는 mRNA로부터 전구체가 합성된 후 post-translational modification에 의해 완전한 SKTI로 변환됨을 알 수 있었다. 또한 SKTI는 대두 종자의 성숙과 함께 발현이 되는 것으로 보아 조직 및 분화 특이성 발현형태를 나타내는 것을 알 수 있었다.

• BMB Reports
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• 제56권5호
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• pp.302-307
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• 2023

Lyn, a tyrosine kinase that is activated by double-stranded DNA-damaging agents, is involved in various signaling pathways, such as proliferation, apoptosis, and DNA repair. Ribosomal protein S3 (RpS3) is involved in protein biosynthesis as a component of the ribosome complex and possesses endonuclease activity to repair damaged DNA. Herein, we demonstrated that rpS3 and Lyn interact with each other, and the phosphorylation of rpS3 by Lyn, causing ribosome heterogeneity, upregulates the translation of p-glycoprotein, which is a gene product of multidrug resistance gene 1. In addition, we found that two different regions of the rpS3 protein are associated with the SH1 and SH3 domains of Lyn. An in vitro immunocomplex kinase assay indicated that the rpS3 protein acts as a substrate for Lyn, which phosphorylates the Y167 residue of rpS3. Furthermore, by adding various kinase inhibitors, we confirmed that the phosphorylation status of rpS3 was regulated by both Lyn and doxorubicin, and the phosphorylation of rpS3 by Lyn increased drug resistance in cells by upregulating p-glycoprotein translation.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1987년도 식물생명공학 심포지움 논문집 Proceedings of Symposia on Plant Biotechnology
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• pp.357-389
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• 1987

In an attempt to clarify the physiological functions of individual isoperoxidases, we have studied enzymatic and immunological properties as well as cellular distribution of isoperoxidases from tobacco callus and Korean radish. The gene expression patterns of isoperoxidases in shoot and non-shoot-forming tobbaco callus were also examined by rabbit reticulocyte lysatein vitro translation system. These results indicate that fraction of translatable poly(A)-isoperoxidase mRNA was increased considerably in shoots. At the present time, at least 6-7 isoperoxidases could be detected from the translation mixture of total cellular RNA, among which only one cell wall localized anodic isoperoxidase (named A3) mRNA was bimorphic mRNA. These data suggest the possible regulation of peroxidase activity during shoot formation by altering the polyadenylation state of mRNA. In case of Korean radish seedlings, poly(A)- peroxidase mRNA were also increased depending upon aging.

• Journal of Plant Biology
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• 제32권1호
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• pp.51-65
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• 1989

Glycinin is the major storage protein in soybean. It has been known that a molecule of glycinin is composed of 6 subunits, each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one (NW 39K and 19K, respectively). To study the molecular origin and the relationship of glycinin subunit polypeptides, antibodies against A-and B-polypeptide were obtained by immunizing rabbits with either of the antigens purified by gel filtration and preparative electrophoresis. Each antibody was not only specific for its own antigen polypeptide in soybeans but also recoginzed the precursor which was synthesized in vivo and in vitro. The polyadenylated mRNAs were isolated from immature seeds and leaves and were translated in vitro using wheat germ extract. One of the seed-specific translation products. MW 60K, was identified to be the precursor of glycinin subunit by immunoprecipitation with antibodies against glycinin A- and B-polypeptide. Mature A- and B-polypeptides were not detected in the translte in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield A- and B-polypeptides which from a glycinin subunit in the cell. Glycinin genes were expressed with the maturation of soybean seeds in a tissue-specific and developmental stage-specific manner.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.311-315
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• 2002

By the in vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis and in vitro coupled transcription/translation with Escherichia coli S30 extract, S65 and E222 of green fluorescent protein of Aequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. The in vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.235-235
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• 2005