scholarly journals Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

2012 ◽  
Vol 23 (23) ◽  
pp. 4543-4551 ◽  
Author(s):  
Yusuke Ohno ◽  
Atsushi Kashio ◽  
Ren Ogata ◽  
Akihiro Ishitomi ◽  
Yuki Yamazaki ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Protein Localization ◽  
Expression System ◽  
Integral Membrane Proteins ◽  
Yeast Expression ◽  
Class Iii ◽  
Substrate Specificities ◽  
Yeast Expression System ◽  
Rich Domain ◽  
Dhhc Protein

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins.

scholarly journals Discovery and evaluation of a novel step in the flavonoid biosynthesis pathway regulated by F3H gene using a yeast expression system

2019 ◽  
Author(s):  
Rahmatullah Jan ◽  
Sajjad Asaf ◽  
Sanjita Paudel ◽  
Sangkyu Lee ◽  
Kyung-Min Kim
Keyword(s):  
Western Blotting ◽  
Rice Plant ◽  
Expression System ◽  
Plant Secondary Metabolites ◽  
Flavonoid Biosynthesis ◽  
Yeast Expression ◽  
List Type ◽  
Biosynthesis Pathway ◽  
Yeast Expression System ◽  
Flavonoid Biosynthesis Pathway

AbstractKaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. These metabolites are produced in low quantities in plants, therefore engineering microbial factory is a favorable strategy for the production of these metabolites. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. The results elucidated a novel step for the first time in kaempferol and quercetin biosynthesis directly from naringenin catalyzed by flavonol 3-hydroxylase (F3H). F3H gene from rice was cloned into pRS42K yeast episomal plasmid (YEP) vector using BamH1 and Xho1 restriction enzymes. We analyzed our target gene activity in engineered and in empty strains. The results were confirmed through TLC followed by Western blotting, nuclear magnetic resonance (NMR), and LC-MS. TLC showed positive results on comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed lack of Oryza sativa flavonol 3-hydroxylase (OsF3H) activity in empty strains while high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for naringenin conversion to both kaempferol and quercetin. These results concluded that rice F3H catalyzes naringenin metabolism via hydroxylation and synthesizes kaempferol and quercetin.HighlightsCurrent study is a discovery of a novel step in flavonoid biosynthesis pathway of rice plant.In this study F3H gene from rice plant was functionally expressed in yeast expression system.Results confirmed that, F3H gene is responsible for the canalization of naringenin and converted into kaempferol and quercetin.The results were confirmed through, western blotting, TLC, HPLC and NMR analysis.

scholarly journals Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System

PLoS ONE ◽  
2009 ◽  
Vol 4 (12) ◽  
pp. e8499 ◽  
Author(s):  
Agata Leszczynska ◽  
Beata Burzynska ◽  
Danuta Plochocka ◽  
Joanna Kaminska ◽  
Magdalena Zimnicka ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Expression System ◽  
Yeast Expression ◽  
Cellular Lipid ◽  
Yeast Expression System

scholarly journals Advantages of yeast-based recombinant protein technology as vaccine products against infectious diseases

2021 ◽  
Vol 913 (1) ◽  
pp. 012099
Author(s):  
C S W Lestari ◽  
G Novientri
Keyword(s):  
Recombinant Protein ◽  
Infectious Diseases ◽  
Recombinant Proteins ◽  
Hansenula Polymorpha ◽  
Expression System ◽  
Cost Effective ◽  
Yeast Expression ◽  
Vaccine Strategy ◽  
Vaccination Programs ◽  
Yeast Expression System

Abstract The yeast expression system is widely used to produce functional recombinant proteins in the biopharmaceutical industry, such as vaccine products. The expression system choices using yeast as the host has many advantages. Various vaccines have been produced commercially using yeast expression systems. This review aims to explore the advantages of the yeast expression system in Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha, which emphasize vaccine products to prevent human infectious diseases. Selection of the appropriate expression system is carried out by identification at the genetic and fermentation levels, taking into account host features, vectors and expression strategies. We also demonstrate the development of a yeast expression system that can produce recombinant proteins, virus-like particles and yeast surface displays as a novel vaccine strategy against infectious diseases. The recombinant protein produced as a vaccine in the yeast system is cost-effective, immunogenic, and safe. In addition, this system has not introduced new microbe variants in nature that will be safe for the environment. Thus, it has the potential to become a commercial product used in vaccination programs to prevent human infectious diseases.

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