Expression and in vitro functional analyses of recombinant Gam1 protein

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Gam1, an early avian adenovirus gene product, is vital for viral replication.
• Gam1 is the first viral protein identified to globally inhibit cellular SUMOylation, a key posttranslational modification.
• Previous characterization of Gam1 lacked sufficient protein purity for structural studies.

Purpose of the Study:

• To clone and express pure, soluble recombinant Gam1 protein for in vitro functional and structural analyses.
• To investigate methods for obtaining soluble Gam1 protein suitable for structural investigations.
• To confirm the SUMOylation inhibitory function of purified Gam1.

Main Methods:

• Cloning and expression of Gam1 in various bacterial systems, including fusion with trigger factor (TF) under a cold shock promoter.
• Optimization of expression conditions, focusing on low-temperature induction and TF chaperone function.
• Purification of soluble Gam1 using sequential chromatography and size exclusion chromatography; analysis by dynamic light scattering.

Main Results:

• Gam1 was insoluble in most tested expression systems but became soluble as a fusion protein with TF.
• Low-temperature induction and TF chaperone activity were critical for enhancing Gam1 solubility.
• Purified monomeric Gam1 confirmed its SUMOylation inhibitory function in vitro.

Conclusions:

• A robust method was established for producing pure, soluble recombinant Gam1 protein.
• The findings provide essential groundwork for future structural studies of Gam1.
• Understanding Gam1 structure will elucidate its role in viral replication and SUMOylation inhibition.