title: Expression and in vitro functional analyses of recombinant Gam1 protein.
abstract: Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication.
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Gustavo A
authors.initials: GA
authors.lastname: Avila
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Ramirez
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Zacariah L
authors.initials: ZL
authors.lastname: Hildenbrand
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Pedro
authors.initials: P
authors.lastname: Jacquez
authors.affiliation: European Institute of Oncology, Department of Experimental Oncology, Via Adamello, 16, 20139 Milan, Italy.
authors.forename: Susanna
authors.initials: S
authors.lastname: Chiocca
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States.
authors.forename: German
authors.initials: G
authors.lastname: Rosas-Acosta
authors.affiliation: Department of Chemistry, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519, United States. Electronic address: cxiao@utep.edu.
authors.forename: Chuan
authors.initials: C
authors.lastname: Xiao
mesh_terms: D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058207:Sumoylation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C105052:CELO protein, adenovirus; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase
keywords: Cold temperature; Gam1; Protein expression; SUMOylation; Trigger factor