Yeast Two-Hybrid System을 이용한 단백질-단백질 결합의 검색

석대현 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.2

There is a great need for general methods to characterize the proteins that contemporary biology makes available. The list of such proteins needing further characterization is glowing and includes proteins already known to be important for specific cellular functions, mutant proteins identified in vivo or made in vitro, and very large numbers of protein being identified by genome projects. The recent success of two-hybrid systems is due to the fact that many cellular functions are carried out by proteins that touch one another. A full understanding of the function of any new protein will require knowledge of the interactions it makes with previously identified proteins. Currently, most new proteins are being identified by large scale sequencing projects. For many of these new proteins the sequence alone sheds little or no light on their function. Two-hybrid technology exploits the fact that transcriptional activators are modular in nature. Two physically distinct functional domains are necessary to get transcription: (1) a DNA binding domain (DBD) that binds to the DNA of the promoter and (2) an activation domain (AD) that binds to the basal transcription apparatus and activates transcription. In the yeast two-hybrid system, the known gene encoding X. is cloned into the "bait" vector. Separately, a second gene (or a library of cDNAs encoding potential interactors). Y. is cloned in frame adjacent to an activation domain of a different transcription factor. Thus, in one strain of yeast, a known protein X is fused to the DNA binding domain of a transcription factor: and in another strain, unknown proteins are fused to the activation domain of another transcription factor. If the one of the unknown proteins combines with X, it will bring the AD over to the DBD, and transcription will be activated.

형광 리포터를 활용한 효모 단백질 잡종 기법 개발

박성균,서수련,황병준,Park, Seong Kyun,Seo, Su Ryeon,Hwang, Byung Joon 한국미생물학회 2019 미생물학회지 Vol.55 No.3

Yeast two-hybrid는 특정 단백질에 대한 상호작용 파트너 단백질의 선별을 위한 방법으로 개발되었다. 하지만 대규모 단백질 상호작용체 분석을 수행하기에 요구되는 노동과 대량의 한천배지 사용에 따른 문제에 의해 널리 사용되지 못하고 있다. 따라서 본 연구에서는 새로운 리포터 시스템을 yeast two-hybrid 방법에 도입하여 fluorescence-activated cell sorting (FACS) 또는 magnetic-activated cell sorting (MACS)를 이용하여 상호작용 파트너 단백질을 포함하는 효모 클론을 손쉽게 선별할 수 있도록 하였다. 새로운 리포터 시스템은 c-myc 항원 결정기가 총 10번 반복되는 형태로 효모 표면에 발현되도록 하였으며, p53과 SV40 T항원을 이용한 실험을 통하여 리포터 단백질의 정상적인 발현을 flow cytometry 분석을 통하여 확인하였다. 따라서, 새로운 리포터 시스템을 도입한 yeast two-hybrid 방법은 대규모 상호작용체 분석을 위해 필요한 노력을 현저히 줄일 수 있을 것으로 기대한다. 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.

효모 이중-중합 시스템을 이용한 G Protein-Coupled Receptor Kinase5(GRK5)의 단백 상호작용 연구

진병철,박태준,김은지,이지은,이정훈,문인규,박정랑,최동주,서봉관 대한순환기학회 2002 Korean Circulation Journal Vol.32 No.7

Genotype–genotype × environment (GGE) biplot analysis of extra-early maturing quality protein maize hybrids for grain yield

Akinyosoye Solomon Tayo 한국작물학회 2022 Journal of crop science and biotechnology Vol.25 No.5

Extra-early-quality protein maize with short maturity period has potential of alleviating protein deficiency and fostering food security among vulnerable people in sub-Saharan Africa. GGE biplot analysis was used to assess forty-five extra-early-quality protein maize hybrids to identify high-yielding and most stable hybrid across environments. The forty-five extra-early-quality protein maize hybrids and two commercial checks were evaluated at three locations (Ibadan, Ile-Ife and Kishi) for two years under rain-fed conditions, making six environments in humid agro-ecologies of Nigeria. Data were collected on grain yield and other agronomic traits, and were subjected to analysis of variance. The results obtained showed that that coefficient of variation ranged from 3.1% (days to anthesis) to 45.6% (grain yield) across locations. The grain yield (kg ha−1) ranged from 1064.72 (Ibadan) to 2175.03 (Kishi), while days to anthesis and days to silking varied from 49.38 and 52.99 (Kishi) to 52.15 and 56.83 (Ibadan), respectively. Genotype, environment and genotype × environment interaction had significant effect on grain yield, where genotype, environment and genotype × environment accounted for 67.0%, 32.6%, 0.12%, respectively of the total variation. This suggests that grain yield of the extra-early-quality protein maize hybrids evaluated was mainly under genetic effect than environmental influence. GGE biplot analysis revealed that hybrid TZEEQI 9 × TZEEQI 16 was adjudged as the most stable and high yielding across locations (ideal genotype), while Ibadan was regarded as ideal environment to discriminate stable hybrids. Therefore, hybrid TZEEQI 9 × TZEEQI 16 can further be tested in multi-locations before recommendation to the farmers in southwestern Nigeria.

Comparative Analyses of Tomato yellow leaf curl virus C4 Protein-Interacting Host Proteins in Healthy and Infected Tomato Tissues

Kim, Namgyu,Kim, Jinnyun,Bang, Bongjun,Kim, Inyoung,Lee, Hyun-Hee,Park, Jungwook,Seo, Young-Su The Korean Society of Plant Pathology 2016 Plant Pathology Journal Vol.32 No.5

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is one of the most important viruses of cultivated tomatoes worldwide, mainly causing yellowing and curling of leaves with stunting in plants. TYLCV causes severe problems in sub-tropical and tropical countries, as well as in Korea. However, the mechanism of TYLCV infection remains unclear, although the function of each viral component has been identified. TYLCV C4 codes for a small protein involved in various cellular functions, including symptom determination, gene silencing, viral movement, and induction of the plant defense response. In this study, through yeast-two hybrid screenings, we identified TYLCV C4-interacting host proteins from both healthy and symptom-exhibiting tomato tissues, to determine the role of TYLCV C4 proteins in the infection processes. Comparative analyses of 28 proteins from healthy tissues and 36 from infected tissues showing interactions with TYLCV C4 indicated that TYLCV C4 mainly interacts with host proteins involved in translation, ubiquitination, and plant defense, and most interacting proteins differed between the two tissues but belong to similar molecular functional categories. Four proteins-two ribosomal proteins, S-adenosyl-L-homocysteine hydrolase, and 14-3-3 family protein-were detected in both tissues. Furthermore, the identified proteins in symptom-exhibiting tissues showed greater involvement in plant defenses. Some are key regulators, such as receptor-like kinases and pathogenesis-related proteins, of plant defenses. Thus, TYLCV C4 may contribute to the suppression of host defense during TYLCV infection and be involved in ubiquitination for viral infection.

Comparative Analyses of Tomato yellow leaf curl virus C4 Protein-Interacting Host Proteins in Healthy and Infected Tomato Tissues

김남규,김진년,방봉준,김인영,이현희,박정욱,서영수 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.5

Tomato yellow leaf curl virus (TYLCV), a member ofthe genus Begomovirus, is one of the most importantviruses of cultivated tomatoes worldwide, mainly causingyellowing and curling of leaves with stunting inplants. TYLCV causes severe problems in sub-tropicaland tropical countries, as well as in Korea. However,the mechanism of TYLCV infection remains unclear,although the function of each viral component hasbeen identified. TYLCV C4 codes for a small proteininvolved in various cellular functions, including symptomdetermination, gene silencing, viral movement,and induction of the plant defense response. In thisstudy, through yeast-two hybrid screenings, we identifiedTYLCV C4-interacting host proteins from bothhealthy and symptom-exhibiting tomato tissues, todetermine the role of TYLCV C4 proteins in the infectionprocesses. Comparative analyses of 28 proteinsfrom healthy tissues and 36 from infected tissues showinginteractions with TYLCV C4 indicated that TYLCVC4 mainly interacts with host proteins involvedin translation, ubiquitination, and plant defense, andmost interacting proteins differed between the twotissues but belong to similar molecular functionalcategories. Four proteins—two ribosomal proteins, Sadenosyl-L-homocysteine hydrolase, and 14-3-3 familyprotein—were detected in both tissues. Furthermore,the identified proteins in symptom-exhibiting tissuesshowed greater involvement in plant defenses. Someare key regulators, such as receptor-like kinases andpathogenesis-related proteins, of plant defenses. Thus,TYLCV C4 may contribute to the suppression of hostdefense during TYLCV infection and be involved inubiquitination for viral infection.

Yeast Two Hybrid Assay를 이용한 Lipocortin-1 결합 단백질 유전자의 분리

이경화,김정우,Lee, Koung-Hoa,Kim, Jung-Woo 배재대학교 자연과학연구소 1997 自然科學論文集 Vol.9 No.1

Glucocorticoid에 의한 항염증 작용의 second messenger로 생각되어지는 annexin superfamily중 하나인 37 kDa의 단백질, lipocortin-1의 작용기작을 이해할 목적으로 in vivo에서 protein-protein interaction을 인식하는 yeast-based genetic assay인 yeast two assay를 통하여 lipocortin-1과 결합하는 단백질 유전자를 분리하여 조사하였다. 이 방법으로 실험을 수행한 결과 분리된 유전자가 human serine proteinase 유전자와 homology가 있는 것으로 밝혀졌다. 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.

Identification of PABPN1 as an Interactive Protein of hnRNP L via Yeast Two-Hybrid Screening

Mie Young Choi 한국유전학회 2008 Genes & Genomics Vol.30 No.5

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the primary pre-mRNA binding proteins found in human cells. The hnRNP L protein is an abundant nucleocytoplasmic shuttling protein. In an attempt to identify the binding partners of hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. One of the cDNA clones was determined to harbor a partial human nuclear poly(A)-binding protein (PABPN1) (GenBank accession number NM_004643). For the first time in this study, hnRNP L is shown to interact specifically with the PABPN1 in the yeast two-hybrid system. The in vitro analysis of the interaction occurring between hnRNP L and PABPN1 was also carried out. The results imply that, even though hnRNP L not able to directly interact with PABPN1 in vitro, protein-protein association between hnRNP L and PABPN1 can be formed by protein-RNA-protein interactions.

Langerhans cell protein 1 (LCP1) binds to PNUTS in the nucleus: implications for this complex in transcriptional regulation

이신정,Jun-Ki Lee,Yong-Sun Maeng,김영명,권영근 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.3

Protein phosphatase-1 (PP1) nuclear targeting subunit (PNUTS), also called PP1R10, p99, or CAT 53 was originally isolated as a mammalian nuclear PP1-binding protein. In this study, we performed yeast two-hybrid screens to identify PNUTS-interacting proteins. Here, we report that LCP1 (epidermal Langerhans cell protein 1), a novel member of the HMG-box protein family, binds tightly to PNUTS. Co-immunoprecipitation of deletion constructs revealed that the C-terminus of LCP1 is sufficient for the interaction with an N-terminal region of PNUTS that is distinct from its PP1-binding domain. Furthermore, immunofluorescence studies showed that a subpopulation of LCP1 co-localizes with PNUTS in nuclear speckles. Importantly, we found that the N-terminus of LCP1 has a strong trans-activation activity in a GAL4-based heterologous transcription assay. The transcriptional activity of LCP1 is markedly suppressed by its interaction with PNUTS, in a PP1-independent manner. These findings suggest that the coordinated spatial and temporal regulation of LCP1 and PNUTS may be a novel mechanism to control the expression of genes that are critical for certain physiological and pathological processes. Protein phosphatase-1 (PP1) nuclear targeting subunit (PNUTS), also called PP1R10, p99, or CAT 53 was originally isolated as a mammalian nuclear PP1-binding protein. In this study, we performed yeast two-hybrid screens to identify PNUTS-interacting proteins. Here, we report that LCP1 (epidermal Langerhans cell protein 1), a novel member of the HMG-box protein family, binds tightly to PNUTS. Co-immunoprecipitation of deletion constructs revealed that the C-terminus of LCP1 is sufficient for the interaction with an N-terminal region of PNUTS that is distinct from its PP1-binding domain. Furthermore, immunofluorescence studies showed that a subpopulation of LCP1 co-localizes with PNUTS in nuclear speckles. Importantly, we found that the N-terminus of LCP1 has a strong trans-activation activity in a GAL4-based heterologous transcription assay. The transcriptional activity of LCP1 is markedly suppressed by its interaction with PNUTS, in a PP1-independent manner. These findings suggest that the coordinated spatial and temporal regulation of LCP1 and PNUTS may be a novel mechanism to control the expression of genes that are critical for certain physiological and pathological processes.

Protein-directed assembly of cobalt phosphate hybrid nanoflowers

Kim, K.H.,Jeong, J.M.,Lee, S.J.,Choi, B.G.,Lee, K.G. Academic Press 2016 JOURNAL OF COLLOID AND INTERFACE SCIENCE - Vol.484 No.-

The understanding and controlling of biomimetic hybrid materials are a key objective in bio-nanotechnology, materials chemistry, and colloid science fields. Biomaterials, such as, enzyme, DNA, RNA, and proteins have become important templates for the construction of inorganic-organic hybrid nanoflowers. From this perspective, we present a simple approach to synthesize protein and metal hybrid flower-like structure using bovine serum albumin (BSA) and cobalt phosphate, and the results of our study on the formation mechanism involved. The time dependent growing stage and formation mechanism were analyzed by electron microscopes and spectroscopic techniques. The protein-directed assembly method for preparation of hybrid nanoflowers described in this work could be used to fabricate other bio-metal hybrid materials with possible applications in biosensors, bioanalytical devices, and industrial biocatalyst fields.