Construction and Characterization of the NS3 Protein of Bluetongue Virus as A Bait for Application in Yeast Two-hybrid System

2021 ◽  
Author(s):  
Ashwini R. Chaple ◽  
Gaurav K. Sharma ◽  
Udit Joshi ◽  
Dhanavelu Muthuchelvan ◽  
Pallavi Deol ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bluetongue Virus ◽  
Clinical Manifestations ◽  
Small Ruminants ◽  
Decisive Role ◽  
Viral Disease ◽  
Yeast Two Hybrid ◽  
Multifunctional Protein ◽  
Ns3 Protein ◽  
Two Hybrid

Background: Bluetongue is an arthropod transmitted viral disease causing range of clinical manifestations in small ruminants. The causative agent, Bluetongue virus (BTV) possesses several structural (SPs) and non-structural proteins (NSPs) associated with its morphogenesis and virulence. NS3, a multifunctional protein of BTV execute a decisive role in virus pathogenesis and release. In the present study, the NS3 protein of BTV was cloned into a pGBKT7-BD vector as bait and characterized for its expression. The suitability of NS3 as a bait to screen interactions with host proteins was also assessed. Methods: The NS3 gene was cloned in the pGBKT7-BD vector to generate the bait plasmid. The recombinant pGBKT7-NS3 bait was sequence confirmed and characterized for auto-activation, toxicity and expression. Thereafter, the bait was mated with yeast two-hybrid (Y2H) cDNA library and screened for the host-BTV protein-protein interactions (PPI). Conclusion: The constructed bait was found suitable for mating, however, no protein hits appeared on forward library screening. The sequence analysis revealed the presence of the transmembrane domains (TM) within the NS3 sequence that may have resulted in the failure of protein interaction studies in Y2H.

20. Discovery of Novel Proteins Involved the Insulin/IGF-1 Signalling Pathway

2018 ◽  
Author(s):  
David (Wen Xiao) Wei
Keyword(s):  
Protein Interactions ◽  
Mutant Form ◽  
Yeast Two Hybrid ◽  
C Elegans ◽  
Insulin Growth Factor ◽  
Novel Proteins ◽  
Similar Phenotype ◽  
Two Hybrid ◽  
Hsp90 Protein ◽  
Novel Protein

The insulin/insulin growth factor-1 (IGF-1) signalling (IIS) pathway plays a key role in metabolism, growth and development. Though research has elucidated aspects of this pathway, it is not fully characterized or understood. A better understanding of the pathway will give insight into related diseases such as cancer. To discover novel proteins involved in the IIS pathway, the C. elegans worm was used due to the homology its insulin/IGF-1 receptor shares with that of humans.  To identify novel protein interactions with the insulin/IGF-1 receptor, we performed a yeast two-hybrid screen using a library of worm proteins. We found several separate interactions with the worm homolog of the HSP90 protein. To support the involvement of HSP90 in the IIS pathway, we studied the phenotypes of worm strains with a mutant form of HSP90. They showed a similar phenotype to those that have a mutant form of the insulin/IGF-1 receptor, inappropriately entering a developmental stage known as dauer. This strongly suggests the involvement of HSP90 in the IIS pathway. Based on previous research, we hypothesized the interaction between HSP90 and the insulin/IGF-1 receptor may allow it to bind other proteins. Thus, we performed a modified yeast two-hybrid screen to identify proteins which interact with the receptor in the presence of HSP90. The screen identified 15 interactions, many more than with the insulin/IGF-1 receptor alone, supporting this hypothesis. Overall, we provide evidence of a novel interaction with insulin/IGF-1 receptor, suggesting HSP90 may be a potential target for developing therapies for IIS pathway related diseases.

scholarly journals A Field-Proven Yeast Two-Hybrid Protocol Used to Identify Coronavirus–Host Protein–Protein Interactions

2015 ◽  
pp. 213-229 ◽  
Author(s):  
Pierre-Olivier Vidalain ◽  
Yves Jacob ◽  
Marne C. Hagemeijer ◽  
Louis M. Jones ◽  
Grégory Neveu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Host Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Analysis of Vaccinia Virus−Host Protein−Protein Interactions: Validations of Yeast Two-Hybrid Screenings

2009 ◽  
Vol 8 (9) ◽  
pp. 4311-4318 ◽  
Author(s):  
Leiliang Zhang ◽  
Nancy Y. Villa ◽  
Masmudur M. Rahman ◽  
Sherin Smallwood ◽  
Donna Shattuck ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Protein Interactions ◽  
Host Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

Protein interactions of fatty acid synthase II

2000 ◽  
Vol 28 (6) ◽  
pp. 615-616 ◽  
Author(s):  
G. Honeyman ◽  
T. Fawcett
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Hybrid Approach ◽  
Carrier Protein ◽  
Yeast Two Hybrid ◽  
Two Hybrid

We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components. Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus.

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