• Title/Summary/Keyword: Yeast two-hybrid

Search Result 232, Processing Time 0.021 seconds

Isolation of the Gene for Lipocortin-1 Binding Protein Using Yeast Two Hybrid Assay (Yeast Two Hybrid Assay를 이용한 Lipocortin-1 결합 단백질 유전자의 분리)

  • Lee, Koung-Hoa;Kim, Jung-Woo
    • The Journal of Natural Sciences
    • /
    • v.9 no.1
    • /
    • pp.25-29
    • /
    • 1997
  • To study the mechanism of lipocortin-1, the 37 kDa protein, one of the annxin superfamily thought to be a second messenger during the Glucocorticoid dependent anti-inflammatory action, the gene for lipocortin-1 binding protein was isolated using the yeast two hybrid assay, the yeast based genetic assay recognizing the protein-protein interaction. The results showed that this gene has a weak homology to the for the human serine proteinase.

  • PDF

Yeast two-hybrid assay with fluorescence reporter (형광 리포터를 활용한 효모 단백질 잡종 기법 개발)

  • Park, Seong Kyun;Seo, Su Ryeon;Hwang, Byung Joon
    • Korean Journal of Microbiology
    • /
    • v.55 no.3
    • /
    • pp.199-205
    • /
    • 2019
  • Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

Screening and Analysis for cTPx II-Interacting Protein Using Yeast Wo-hybrid System (Yeast Two-hybrid System을 이용한 cTPx II 결합단백질 탐색 및 분석)

  • Kim. Il-Han;Oh, Young-Mee;Cha, Mee-Kyung
    • The Journal of Natural Sciences
    • /
    • v.15 no.1
    • /
    • pp.79-88
    • /
    • 2005
  • There are five isoforms of thiol peroxidase in yeast. Each isoform was named after its subcellular localization such as cytoplasmic TPx I, cTPx II, cTPx III, mitochondrial TPx (mTPx), and nuclear TPx (nTPx). Recently, we reported that unlike other TPx null mutants, cTPx IInull mutant showed a slow-growth phenotype. This observation suggests that cTPx II might be involved in yeast cell growth. In this study, for a first step toward to investigate the physiological function of cTPx II in yeast, we have identified a novel interaction between cTPx II and various proteins by using the yeast two-hybrid system.

  • PDF

Detection of Ref-1 (Redox factor-1) Interacting Protein Using the Yeast Two-hybrid System (Yeast two-hybrid system을 이용한 Ref-1 (redox factor-1) 결합 단백질의 분리 및 동정)

  • 이수복;김규원;배문경;배명호;정주원;안미영;김영진
    • Journal of Life Science
    • /
    • v.14 no.1
    • /
    • pp.26-31
    • /
    • 2004
  • Redox factor-1 (Ref-1), known as a redox regulator, controls the DNA binding of AP-1 and is activated in HT29 colon cancer cells by hypoxia in vitro. REF-1 also increases tile DNA binding affinity of Hypoxia-inducible Factor-lalpha$ (HIF-lalpha$), HIF-like Factor (HLF) and early growth response-1 (Egr-1) which induce expression of the genes involved in angiogenesis, so that we speculate that REF-1 may play a role in hypoxia-induced angiogenesis. In this research we tried to detect novel proteins interacting with REF-1 using Yeast two-hybrid system using full-length REF-1 cDNA as bait. As result of such screening we detected 3 positive clones. DNA sequencing and GeneBank search revealed that one of the clones contained the same sequences as M.musculus cDNA for tioredoxin.

Isolation of the Gene for HIV-1 gp41 Interacting Protein (HIV gp41의 세포내 부분과 상호작용하는 단백질 유전자의 분리)

  • Kim, Eun-Mi;Kim, Jung-Woo
    • The Journal of Natural Sciences
    • /
    • v.10 no.1
    • /
    • pp.27-32
    • /
    • 1998
  • To find the interacting protein with the cytoplasmic domain of HIV-1 gp41, the yeast two hybrid system was used for the expression cloning. Among the $1.4 \times 10^6 colonies, 20 colonies were selected as the final candidate for the interacting protein gene. The nucleotide sequencing revealed three kinds of protein, acidic ribosomal protein P0, beta tubulin, alpha catenin. These proteins interacted with the gp41 specifically in yeast system.

  • PDF

Cloning of hnRNP E1 cDNA via yeast two-hybrid system and a study on protein-protein interaction between hnRNP E1 and hnRNP K (이스트 two-hybrid 시스템을 이용한 hnRNP E1 cDNA의 클로닝과 hnRNP E1-hnRNP K 상호결합에 대한 연구)

  • Choi, Mie-Young
    • Journal of the Korea Academia-Industrial cooperation Society
    • /
    • v.9 no.6
    • /
    • pp.1795-1799
    • /
    • 2008
  • The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a component of hnRNP complexes. This protein binds strongly to cytidine-rich RNA/DNA sequences. It is a nucleocytoplasmic shuttling protein. To investigate the functions of hnRNP K, I searched for hnRNP K-interacting proteins in HeLa cDNA library using a yeast two-hybrid screening system. One of the cDNA clones is identical to human hnRNP E1 (poly(rC) binding protein 1) cDNA (GenBank accession number XM_031585). In this study, hnRNP K is shown to specifically interact with hnRNP E1 in yeast two-hybrid system and in vitro biochemical assay.

Activated Phenoloxidase Interacts with A Novel Glycine-rich Protein on the Yeast Two-hybrid System

  • Lee, Sun-Woo;Lee, Hyun-Seong;Kim, Eun-Jun;Yoo, Mi-Ae;Lee, Bok-Luel
    • BMB Reports
    • /
    • v.34 no.1
    • /
    • pp.15-20
    • /
    • 2001
  • One of the innate immune reactions in invertebrates is the pro-phenoloxidase (pro-PO) activation system that is involved in the generation of superoxide, melanin synthesis, and the subsequent sequestration of foreign matter entering the hemocoel of the invertebrates. However, the molecular mechanism of this biological reaction is still obscure. To expand our understanding of the biological roles of the pro-PO activation system in invertebrates, we performed a yeast two-hybrid screening by using three regions of pro-PO as bait and a yeast two-hybrid cDNA library from Tenebrio molitor larvae as prey We isolated a novel partial cDNA clone that encodes a glycine-rich protein that interacted with the active phenoloxidase (termed phenoloxidase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a C-terminal glycine-rich domain. The C-terminal glycine-rich domain showed sequential homology with those of insect antifungal proteins. Also, the yeast two-hybrid screen in a reverse orientation (using POIP as bait) yielded PO, suggesting that the PO-POIP interaction is specific. By using a 315 bP PCR fragment of the N-terminal unique region of POIP, we cloned the full-length cDNA of POIP from the Tenebruo cDNA library constructed by using E. coli injected larvae. The interaction analysis between PO, and a truncated fragment lacking the N-terminal unique region of POIP, indicated that the N-terminal unique region is necessary for interaction between PO and POIP. The expression level of the POIP mRNA is increased by bacterial injection into T. molitor larvae. This suggests that POIP might be engaged in the humoral defense reaction.

  • PDF

Use of the Yeast 1.5-Hybrid System to Detect DNA-Protein-Protein Interaction

  • Kim, Sook-Kyung;Han, Jin-Hee
    • Journal of Microbiology
    • /
    • v.38 no.2
    • /
    • pp.113-116
    • /
    • 2000
  • Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find and additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The results indicates that this system is a promising one, capable of selecting an interacting component.

  • PDF

Interaction of a Kinesin Superfamily Protein 1A (KIF1A) with Calmodulin

  • Seog, Dae-Hyun
    • Journal of Life Science
    • /
    • v.12 no.2
    • /
    • pp.43-46
    • /
    • 2002
  • Kinesin Superfamily Protein 1A (KIF1A) is an anterograde monomeric motor transporting a subset of synaptic vesicle precursors and plays an important role in neuronal function and survival. Here, f have used the yeast two-hybrid system to identify the proteins that interacts with the tail region of KIF1A. Calmodulin was found to interact specifically with the tail region of KIF1A. Calmodulin regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. KIF1A interacts with calmodulin in the yeast two-hybrid assay, which is proved by immunoprecipitation with calmodulin in brain fraction. These results indicate that KIF1A is associated with calmodulin, suggesting that calmodulin may be a key role in the regulation of anterograde transport of synaptic 1 vesicle precursors.

  • PDF