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PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait

Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted pro...

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Main Authors: Nur Athirah Abd Hamid,, Ismanizan Ismail,
Format: Article
Language:English
Published: Penerbit Universiti Kebangsaan Malaysia 2018
Online Access:http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf
http://journalarticle.ukm.my/12916/
http://www.ukm.my/jsm/malay_journals/jilid47bil12_2018/KandunganJilid47Bil12_2018.html
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spelling my-ukm.journal.129162019-05-15T10:58:02Z http://journalarticle.ukm.my/12916/ PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait Nur Athirah Abd Hamid, Ismanizan Ismail, Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS. A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat (FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system (Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins. Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique. Those genes may code for potential interacting proteins that needs further identification and confirmation. Penerbit Universiti Kebangsaan Malaysia 2018-12 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf Nur Athirah Abd Hamid, and Ismanizan Ismail, (2018) PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait. Sains Malaysiana, 47 (12). pp. 2961-2968. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid47bil12_2018/KandunganJilid47Bil12_2018.html
institution Universiti Kebangsaan Malaysia
building Perpustakaan Tun Sri Lanang Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Kebangsaan Malaysia
content_source UKM Journal Article Repository
url_provider http://journalarticle.ukm.my/
language English
description Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS. A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat (FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system (Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins. Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique. Those genes may code for potential interacting proteins that needs further identification and confirmation.
format Article
author Nur Athirah Abd Hamid,
Ismanizan Ismail,
spellingShingle Nur Athirah Abd Hamid,
Ismanizan Ismail,
PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
author_facet Nur Athirah Abd Hamid,
Ismanizan Ismail,
author_sort Nur Athirah Abd Hamid,
title PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_short PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_full PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_fullStr PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_full_unstemmed PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_sort peg-4000 increased the mating efficiency of yeast-two hybrid screening process using pmf-box1 as bait
publisher Penerbit Universiti Kebangsaan Malaysia
publishDate 2018
url http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf
http://journalarticle.ukm.my/12916/
http://www.ukm.my/jsm/malay_journals/jilid47bil12_2018/KandunganJilid47Bil12_2018.html
_version_ 1643738926261731328
score 13.211869

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