Expression Profiling of Herpesvirus and Vaccinia Virus Proteins Using a High-Throughput Baculovirus Screening System Mian Gao, †,# Nicole Brufatto, †,⊥ Tricia Chen, † Laura Lea Murley, † Rosanne Thalakada, † Megan Domagala, † Bryan Beattie, † Daniel Mamelak, †,& Vicki Athanasopoulos, ‡,∧ David Johnson, § Grant McFadden, | Christian Burks, †,∇ and Lori Frappier* ,†,‡ Affinium Pharmaceuticals, 100 University Avenue, Toronto, Canada M5J 1V6, Department of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Canada M5S 1A8, Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201-3098, Department of Microbiology and Immunology, University of Western Ontario, and Robarts Research Institute, 1400 Western Road, London, Ontario, Canada N6G 2V4 Received May 10, 2005 We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV- 1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus. Keywords: baculovirus • high-throughput protein expression • Epstein-Barr virus • herpes simplex virus • cytomegalovirus • vaccinia Introduction Herpesviruses and poxviruses have large double stranded DNA genomes, 120-280 Kb in length, that each encode between 80 and 200 proteins. This complex network of proteins results in a variety of effects on the host cell and ensures efficient proliferation of the virus. While the functions of a small subset of these proteins are reasonably well understood, there is little or nothing known about the function of many of the proteins and very few high-resolution structures determined for these viral proteins. Studies on these proteins have been hampered by lack of reagents (purified proteins and antibodies) and difficulty in generating many of the proteins in E. coli. As a first step toward generating viral proteins for subsequent studies, we have made baculoviruses encoding the majority of proteins in the genomes of three different human herpesvi- ruses, herpes simplex type 1 (HSV-1), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), and for 78 proteins from the vaccinia poxvirus (for reviews of these viruses see ref 1). The three herpesviruses selected are representative of the three classes of herpesviruses and differ considerably in their host cell interactions and pathology. HSV-1 is a common alpha- herpesvirus that causes oral herpes, has dramatic effects on shutting down host cell processes and is closely related in protein sequence and composition to the HSV-2 genital herpes virus. CMV is a very common beta-herpesvirus that is usually asymptomatic, but causes a variety of illnesses in immuno- compromised patients and can cause neurological problems in babies infected in utero. EBV is a gamma-herpesvirus that infects most people worldwide. It is the causative agent of mononucleosis and predisposes the host to a variety of cancers due to its ability to immortalize latently infected cells. The orthopoxvirus, vaccinia virus, is the most widely studied of the poxviruses. It infects humans and other species and is a close relative of variola, the causative agent of smallpox. To generate these proteins in a timely fashion, we have developed a high throughput system for generating recombi- nant bacmids and baculoviruses and screening the baculovi- ruses for the expression, solubility, and ease of purification of the encoded protein. The results for 337 viral proteins are presented and compared to E. coli expression. Experimental Section Cloning. Viral genes were PCR amplified from samples of the complete viral DNA genome. For HSV-1, extracts prepared from cells infected with HSV-1 strain 17 were used as the PCR template (prepared by Dr. Karen Mossman). For EBV, B95-8 cells harboring the B95-8 strain of EBV were induced to produce virus by serum starvation and TPA treatment and cell extracts were used for PCR templates. CMV genes were PCR amplified * To whom correspondence should be addressed: lori.frappier@utoronto.ca. † Affinium Pharmaceuticals. ‡ University of Toronto. § Oregon Health Sciences University. | University of Western Ontario. # Present address: Gene Expression & Protein Biochemistry, Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, NJ 08543. ⊥ Present address: CanReg Inc., 4 Innovation Drive, Dundas, Ontario Canada L9H 7P3. & Present address: Custom Biologics, 1 Marmac Drive, Toronto, Ontario Canada M9W 1E7. ∧ Present address: Research School of Biological Sciences, Sullivan’s Creek Road, The Australian National University, ACT Acton 2601 Australia. ∇ Present address: Ontario Genomics Institute, 149 College Street, suite 500, Toronto, Ontario Canada M5T1P5. 10.1021/pr050137u CCC: $30.25  2005 American Chemical Society Journal of Proteome Research 2005, 4, 2225-2235 2225 Published on Web 09/17/2005