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

Nur Athirah Abd Hamid, Ismanizan Ismail

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)2961-2968
Number of pages8
JournalSains Malaysiana
Volume47
Issue number12
DOIs
Publication statusPublished - 1 Dec 2018

Fingerprint

two hybrid system techniques
conception rate
polyethylene glycol
baits
yeasts
screening
F-box proteins
ubiquitin
proteins
ubiquitin-protein ligase
genes
protein degradation
toxicity testing
reporter genes
gel electrophoresis
abscisic acid
RNA
sampling
degradation
fish

Keywords

  • PmF-box1
  • Polyethylene glycol-4000
  • Yeast-two hybrid

ASJC Scopus subject areas

  • General

Cite this

PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as Bait. / Hamid, Nur Athirah Abd; Ismail, Ismanizan.

In: Sains Malaysiana, Vol. 47, No. 12, 01.12.2018, p. 2961-2968.

Research output: Contribution to journalArticle

@article{4909b73e1c834457b421823ad0d5b2be,
title = "PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as Bait",
abstract = "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.",
keywords = "PmF-box1, Polyethylene glycol-4000, Yeast-two hybrid",
author = "Hamid, {Nur Athirah Abd} and Ismanizan Ismail",
year = "2018",
month = "12",
day = "1",
doi = "10.17576/jsm-2018-4712-04",
language = "English",
volume = "47",
pages = "2961--2968",
journal = "Sains Malaysiana",
issn = "0126-6039",
publisher = "Penerbit Universiti Kebangsaan Malaysia",
number = "12",

}

TY - JOUR

T1 - PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as Bait

AU - Hamid, Nur Athirah Abd

AU - Ismail, Ismanizan

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

AB - 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.

KW - PmF-box1

KW - Polyethylene glycol-4000

KW - Yeast-two hybrid

UR - http://www.scopus.com/inward/record.url?scp=85060850655&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85060850655&partnerID=8YFLogxK

U2 - 10.17576/jsm-2018-4712-04

DO - 10.17576/jsm-2018-4712-04

M3 - Article

VL - 47

SP - 2961

EP - 2968

JO - Sains Malaysiana

JF - Sains Malaysiana

SN - 0126-6039

IS - 12

ER -