Expression Profiling of Herpesvirus and Vaccinia Virus Proteins Using

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 (HSV1, 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 herpesviruses, 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 * To whom correspondence should be addressed: [email protected]. † 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

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 alphaherpesvirus 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 immunocompromised 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 recombinant bacmids and baculoviruses and screening the baculoviruses 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 Journal of Proteome Research 2005, 4, 2225-2235

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research articles from viral DNA prepared from strains AD169 and Towne. For vaccinia virus, extracts prepared from cells infected with the WR strain of the virus were used as PCR templates. The EBV genes BZLF1 and EBNA3B were PCR amplified from cloned cDNAs supplied by Dr. George Miller and Dr. Elliott Kieff, respectively. Oligonucleotides were designed to correspond to genes defined by NCBI entries for each viral strain and specific restriction sights were included to enable cloning between unique sites in modified pFastBac and pET15b plasmids. In both cases, expressed proteins are fused at the N-terminus to a hexahistidine purification tag. High-Throughput Production of Recombinant Bacmids. The recombinant donor plasmids bearing viral genes were prepared using QIAprep 96 Turbo miniprep kit (Qiagen) and stored in a 96-well PCR plate. Fifty µL of E. coli competent DH10BAC cells (Invitrogen) were dispensed into each well of four 24-well blocks (Qiagen) using a multichannel pipet. Three microliters of recombinant donor plasmids were transferred from the PCR plate into the 24-well blocks using a multichannel pipet. The blocks were incubated on ice for 30 min followed by 45 s in a 42 °C water bath and 2 min on ice. SOC (0.9 mL) was added to each well and the blocks were incubated at 37 °C for 5 h at 240 rpm. One µL of the culture from each well was spread on LB plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 200 µg/mL halogenated indolyl-β-galactoside (bluo-gal) and 40 µg/mL isopropylβ-D-thiogalactopyranoside (IPTG). Recombinant bacmids were selected at 37 °C for 40 h. One well-isolated white colony was picked from each plate and cultured in 3 mL of LB with the appropriate antibiotics overnight in 4 24-well blocks. Recombinant bacmids were prepared using Montage BAC96 miniprep kit (Millipore) according to the instructions provided by the manufacturer. The purified recombinant bacmids were suspended in 50 µL of storage buffer and transferred into a PCR plate. High-Throughput Production of Recombinant Baculovirus. Spodoptera frugiperda (Sf9) cells (Invitrogen) were cultured in suspension in HyQ-SFX-Insect serum free medium (Hyclone). One milliliter of Sf9 cells at 2 × 105 cells/ml was seeded into each well of four 24-well plates and incubated at room temperature before transfection. Six microliters of recombinant bacmids were mixed with 50 µL of HyQ-SFX-Insect serum free medium in four 24-well plates. Then, 50 µL of the same medium containing 3 µL of Cellfectin reagent (Invitrogen) was added to each well. After incubation at room temperature for 30 to 45 min, 200 µL of medium was added to each well and gently mixed. The medium was aspirated from the cells in the 24-well plates and the diluted lipid-DNA complexes were overlaid onto the cells. The lipid-DNA complexes were incubated with the cells for 5 h at 27 °C, then replaced with 0.7 mL of Grace’s medium (Invitrogen) with 10% FBS. The culture media (P1 viruses) were collected 88 to 96 h after transfection and stored in a 96 deep well plate. High-Throughput Test Expression in Insect Cells. Trichoplusia ni (High-5) cells (Invitrogen) were cultured in suspension in HyQ-SFX-Insect serum free medium. Three ml of High-5 cells at 2 × 106 cells/ml were dispensed into each well of four 24-well blocks. Sixty microliters of the P1 viruses were added to corresponding wells and the blocks were cultured at 27 °C for 48 h with shaking at 240 rpm. Infected cells were harvested and suspended in purification buffer (50 mM Hepes pH 7.5, 0.5 M NaCl, 5% glycerol) with 5 mM imidazole, benzonase nuclease (EMD Biosciences), and protease inhibitor cocktail 2226

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(Roche Diagnostics). The cell suspensions were split equally and lysed in 96 deep well blocks under native (0.5% NP-40) and denatured (0.5% NP-40 and 6 M urea) conditions. After centrifugation, the cleared lysates were transferred into new deep well blocks and incubated with Ni-NTA resin (Qiagen). After binding, the Ni-NTA resin was transferred into 96-well filter plates and washed three times with 0.3 mL purification buffer containing 30 mM imidazole with (denatured samples) or without (nondenatured samples) 6 M urea. Proteins bound to the Ni-NTA resin were eluted with purification buffer containing 0.5 M imidazole with (denatured samples) or without (nondenatured samples) 6 M urea. Proteins purified from 0.1 mL of culture under native and denatured conditions were analyzed on 26-well Criterion SDS-PAGE (Bio-Rad Laboratories) to determine the expression and solubility of the protein. In some cases, eluted proteins were trypsinized and analyzed by MALDI-TOF mass spectrometry to determine protein expression, solubility, and identity. Test Expression in E. coli. Viral proteins were expressed with N-terminal hexahistidine tags in small scale cultures of a E. coli BL21 Gold (DE3) (Stratagene) containing an extra plasmid that expresses three rare tRNAs (AGA and AGG for Arg and ATA for Ile).2 Cultures (4 mL) were grown in Luria Broth to an optical density of 0.6, at which time IPTG was added to 0.5 mM. For CMV, EBV, and HSV proteins, cultures were induced overnight at 15 °C. For vaccinia virus proteins, cultures were induced at 37 °C for 3 h. Cells from two 1.5 mL samples of the culture were harvested and used to generate total and soluble cell extracts. Total cell extracts were generated by resuspending the cell pellet in 200 µL of a 50:50 mixture of PBS (130 mM NaCl, 6 mM Na2HPO4, 6 mM KH2PO4 pH to 6.9) and gel sample buffer (100 mM DTT, 2%SDS, 80 mM Tris pH 6.8, 0.006% Bromophenol Blue, 15% glycerol), then boiling and analyzing the samples by SDS-PAGE. Soluble cell extracts were generated by flash freezing the cell pellets at -80 °C, then thawing, resuspending in 160 µL 500 mM NaCl, 18 µL 10× Bugbuster (Qiagen) and 1 µL Benzonase and incubating at room temperature for 30 min. Samples were then clarified by centrifugation at 4 °C and the resulting supernatant was analyzed by SDS-PAGE. After Coomassie staining, gels were analyzed visually for low, medium or high-density bands at the expected molecular weight for the protein.

Results and Discussion Optimization of High-Throughput Baculovirus Production Procedure. The Bac-To-Bac baculovirus expression system allows rapid and efficient generation of baculovirus. However, most steps in the protocol provided by the supplier involve handling samples individually in test tubes and culture flasks, thus limiting the number of samples that can be dealt with simultaneously. We have made improvements in a number of steps to greatly increase the throughput capacity, as summarized in Figure 1. First, in the transposition step, the replacement of test tubes with 24-well blocks has allowed us to process a plate of cloned DNA rapidly. With no need to label, open, and close individual tubes, the time spent on mixing DNA with competent cells, heat shock, and recovery is greatly reduced. Second, in the recombinant bacmid preparation step, we used 24-well blocks instead of test tubes for overnight culture and adapted the Montage BAC96 miniprep kit from Millipore, which was initially designed for isolating recombinant bacterial artificial chromosomes, to isolate recombinant bacmids in a 96-well format. Third, in the transfection step,

Expression Profiling of Herpesvirus and Vaccinia Virus

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Figure 1. Schematic representation of the high throughput baculovirus system. Flowchart of bacmid and baculovirus generation and test expression is shown.

Figure 2. Test expression of viral proteins in insect cells. Baculoviruses encoding the indicated viral proteins were generated in duplicate and used to infect insect cells. The expression and solubility of the protein was examined using the high-throughput system described in the Experimental section. Proteins were purified from 1.5 to 3.0 mL of culture under native (S) or denaturing (T) conditions to give soluble and total protein samples, respectively, and aliquots equivalent to 0.1 mL of culture were analyzed by SDS-PAGE and Coomassie staining. For each baculovirus, the most prominent band seen corresponds to the expect molecular weight of the expressed viral protein.

by varying the amount of cells and the volume of lipid-DNA complex, we are able to obtain conditions where sufficient viral titers were generated from cells seeded in 24-well plates to allow subsequent analysis of protein expression. The preparation of lipid-DNA complexes in 24-well plates also greatly reduced the time in setting up the transfections. With all of these changes, each person can comfortably process two 96well plates of clones simultaneously.

Optimization of High Throughput Baculovirus Test Expression Procedure. Traditional small scale protein expression profiling involves analysis of soluble and insoluble fractions of infected cells by immunoblotting. However, setting up the infection in 6-well plates and the preparation of soluble and insoluble fractions from infected cells is very labor intensive and time-consuming. In addition, the results of immunoblotting do not give information on whether the protein expressed Journal of Proteome Research • Vol. 4, No. 6, 2005 2227

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Table 1. Expression and Solubility of HSV-1 Proteins in Insect Cells ORF/protein name

ORF O ORF P RL1 UL2 UL3 UL4 UL6 UL7 UL8 UL8.5 UL9 UL9.5 UL12 UL13 UL14 UL15.5 UL16 UL17 UL18 UL19 UL20.5 UL21 UL23 UL24 UL25 UL26 UL26.5 UL27.5 UL29 UL31 UL32 UL33 UL34 UL35 UL37 UL38 UL40 UL41 UL42 UL43.5 UL45 UL47 UL48 UL49 UL49A UL50 UL51 UL52 UL54 UL55 UL56 US1 US1.5 US2 US3 US8A US9 US10 US12

accession no.

HHVONE061.2a HHVONE061.1a GI:9629379/ GI:9629440 GI:9629382 GI:9629383 GI:9629384 GI:9629386 GI:9629387 GI:9629388 aa365-850 of GI:9629389 GI:9629389 HHVONE012.1a GI:9629392 GI:9629393 GI:9629394 aa443-735 of GI:9629397 GI:962935 GI:9629396 GI:9629398 GI:9629399 HHVONE022.1a GI:9629401 GI:9629403 GI:9629404 GI:9629405 GI:9629406 GI:9629407 HHVONE029.1a GI:9629410 GI:9629412 GI:9629413 GI:9629414 GI:9629415 GI:9629416 GI:9629418 GI:9629419 GI:9629421 GI:9629422 GI:9629423 HHVONE046.1a GI:9629426 GI:9629428 GI:9629429 GI:9629430 GI:9629431 GI:9629432 GI:9629433 GI:9629434 GI:9629436 GI:9629437 GI:9629438 GI:9629442 aa 147-420 of GI:9629442 GI:9629443 GI:9629444 GI:9629450 GI:9629451 GI:9629452 GI:9629454

protein size (kDa, with tag)

expression levela

solubilityb

14 26 28

low very low high

very low very low very low

38 28 24 76 35 82 57

high high low medium medium none low

very low very low none medium none none very low

96 53 70 59 26 34

low high high low high high

low very low none very low low high

43 77 36 151 20 60 43 32 65 69 36 63 130 36 66 17 32 14 338 52 40 57 53 35 20 76 56 34 11 41 28 117 57 23 27 49 33

high medium high very low low medium high very low low very low high medium high very low very low medium medium none very low low high very low high medium none none high high very low high high very low low high very low medium low

medium medium high none none very low high very low very low none none very low high very low very low very low very low none none very low high none high medium none none high high none high high very low very low medium very low low very low

35 55 19 12 36 12

low very low none very low very low high

very low very low none none very low very low

a Gene ID as defined in HSV-1 genome at http://biosphere.lanl.gov/ readonly/stdgen/(no GenBank ID available for this protein). a Expression level was estimated from Coomassie-stained gels of proteins prepared under denaturing conditions. Expression was designated as “high” if expression was greater than 10 mg per liter of insect cells, “medium” if expression was 5 to 10 mg per liter of insect cells, “low” if expression was 1 to 5 mg/L of insect cells, “very low” if expression was less than 1 mg/L of insect cells but detectable in Coomassie-stained gels or by MALDI-TOF mass spectrometry, and “none” if the protein was not detected by either Coomassie-stained gels or mass spectrometry. b Solubility was estimated from Coomassie-stained gels of proteins prepared under nondenaturing conditions. Amounts of proteins indicated are as defined in a.

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is amenable to purification or an accurate indication of the amount of protein expressed. We carried out the test expression of the recombinant baculoviruses encoding hexahistidine tagged proteins in suspension cells grown in 24-well blocks. The growth of the cells in suspension resulted in test expression conditions that are more consistent with large scale growth, which is also done in suspension, and also greatly simplified the collection of infected cells. Various conditions were explored for lysing the collected cells, and NP-40 was found to be the most effective detergent for insect cell lysis. Benzonase nuclease was also included to reduce the viscosity of the lysate and facilitate filtration after incubation of the lysates with nickel resin. Each sample of infected cells harvested from the 24-well blocks was split into two equal parts, where one sample was lysed in the presence of 6 M urea to denature the proteins while the other sample was lysed under native conditions (see Figure 1). The native and denatured samples were incubated separately with nickel resin in 96-well plates, then filtered to separate resin-bound and unbound proteins. Proteins were eluted from the nickel resin with imidazole and analyzed by SDS-PAGE and Coomassie staining (Figure 2). Thus, our test expression procedure mimics that of large-scale protein purification, enabling the prediction of the yield expected from large scale culture. It also allows one to assess the solubility of the expressed protein, since insoluble proteins would be recovered from the resin only in the presence of urea, while soluble proteins would be recovered from the resin under both native and denaturing conditions. The ability to perform the test purifications in a 96-well format greatly increased the throughput of the procedure and allowed for reliable estimations of protein solubility and ease of purification from as little as 1.5 mL of culture. Expression of Viral Proteins in Insect Cells. We have used the high throughput system described above to generate baculoviruses for and test the expression and solubility of 337 proteins from HSV-1, EBV, CMV and vaccinia virus. Viral genes were defined according to the NCBI entries for HSV-1 strain 17 (GI:9629378), EBV strain B98-5 (GI: 9074) and vaccinia virus strain WR (GI: 29692106). For CMV, most of the genes were cloned from strain AD169 according to the NCBI entry GI:59591 but, since this laboratory strain is known to lack a region encoding UL133 to UL151, which is present in the more virulent Towne and Toledo strains, we also included genes found only in Towne and Toledo. In this case, cDNAs were PCR amplified from the Towne virus using sequence information from the Toledo strain at NCBI entry GI:1167917 (the sequence of the Towne strain was not available at the time of cloning). In all cases, genes were PCR amplified from samples containing the viral genomic DNA. Genes that did not yield PCR products on initial attempts were not further pursued. HSV, EBV, and CMV all contain a few open reading frames generated by splicing; these were not included in this study, with the exception of EBV genes BZLF1 and EBNA3B where cloned cDNAs were obtained. After cloning into a baculovirus transfer plasmid that places a hexahistidine tag at the N-terminus of each protein, bacmids and baculoviruses were generated and analyzed in the test expression system. To test the reproducibility of the system, two bacmids and baculoviruses were generated for each protein and the duplicates were individually tested for expression and solubility. In all cases, there was excellent agreement in the level of protein expression and solubility in the duplicate viruses (see examples in Figure 2). For the herpesviruses,

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Expression Profiling of Herpesvirus and Vaccinia Virus Table 2. Expression and Solubility of EBV Proteins in Insect Cells ORF/protein name

BALF1 BALF2 BALF3 BARF1 BARF1 BBLF1 BBLF2 BBLF3 BBLF4 BBRF1 BBRF2 BCRF1 (IL-10) BCRF1 (UL87) BDLF1 BDLF2 BDLF3 BDLF4 BDRF1 BERF1 BFLF2 BFRF1 BFRF2 BFRF3 BGLF1 BGLF3 BGLF4 BGLF5 BGRF1 BILF2 BKRF3 BKRF4 BLLF2 BLLF3 BLRF1 BLRF2 BMLF1 BMRF1 BNLF2a BNLF2b BORF1 BORF2 BRLF1 BRRF1 BRRF2 BSLF1 BSLF2 BSRF1 BTRF1 BVLF1.5(C) BVLF1.5(N) BVRF1 BVRF2 BXLF1 BXRF1 BZLF1 BZLF2 EBNA2 EBNA3A EBNA3B

alternative name/function

bcl-2 homolog major ssDNA binding protein DNA packaging and transport protein, homology to HSV UL28 BARF1 Ribonucleotide reductase, small subunit Myristylated tegument protein DNA helicase-primase component Hypothetical protein helicase Minor capsid protein, Herpesvirus UL6 like UL7 family Viral Interleukin 10 Herpesvirus UL87 family minor capsid protein, HSV VP23 homologue Hypothetical protein Hypothetical protein putative metal binding protein, UL92 family scaffold protein, C terminus of BVRF2 Hypothetical protein Herpesvirus UL31-like protein Herpesvirus virion protein U34 possible capsid protein, UL49 family Gammaherpesvirus capsid protein Viral DNA cleavage/packaging protein Hypothetical protein serine threonine kinase Alkaline exonuclease DNA packaging protein-UL15 family Hypothetical protein uracil DNA glycosylase Hypothetical protein hypothetical protein dUTPase homology to HHV5 UL73 envelope glycoprotein Hypothetical protein SM/EB2 protein minus N-terminal fragment DNA polymerase processivity factor Hypothetical protein hypothetical protein capsid protein, VP19C homologue Ribonucleotide-reductase, large subunit Rta transcriptional activator Na transcription factor homology to HHV8 orf 48, glycoprotein L DNA helicase-primase component N-terminal fragment (5′ exon) of SM/EB2 protein HHV8 orf 55 homolog; homology to DNA helicase-primase component UL52/UL70 Hypothetical protein gp127, C-terminal fragment of BVLF1.5 gp128, N-terminal fragment of BVLF1.5 Capsid associated tegument protein (UL25 family) proteinase/scaffold protein Thymidine kinase UL24 family Zta, ZEBRA gp42 glycoprotein BYRF1, latency nuclear antigen 2 BLRF3, latency nuclear antigen 3A BPLF1, latency nuclear antigen 3B

expression


levela

solubilityb

GI:1334918 GI:9625660 GI:9625659

27 125 88

very low low low

very low low very low

GI:1334917 GI:1334857 GI:1334890 GI:1334888 GI:1334887 GI:1334885 GI:1334884 GI:1632797 GI:59076 GI:9625647 GI:9625645 GI:9625644 GI:1334899 GI:1334897 GI:1334910 GI:5918021 GI:19034032 GI:9625596 GI:1632788 GI:1632789 GI:1334896 GI:1334894 GI:9625636 GI:1334891 GI:1334893 GI:1334911 GI:9625626 GI:1632796 GI:9625613 GI:1334864 GI:1334865 GI:1334866 GI:1632790 GI 330355 GI:1334920 GI:1334919 GI:225105 GI:1334856 GI:1334878 GI:73983 GI:9625623 GI:1334862 GI:5918020 GI:9625609

27 36 11 59 25 92 71 33 22 66 36 48 26 28 38 6 37 40 66 20 57 40 53 55 39 29 31 26 19 33 13 20 51 45 9 14 41 95 69 37 59 100 6 26

low medium none low low very low very low low very low very low high none very low high high low medium very low none medium medium low none very low very low low high low high high very low high very low high medium medium low low very low high low very low medium medium

none medium none very low none none very low none very low very low high none none medium very low low very low very low none none medium very low none none none none very low low very low high none high very low high very low none very low very low very low very low low very low medium very low

GI:1334904 GI:19034033 GI:19034034 GI:1334908 GI:1334909 GI:1334907 GI:1334906 GI:1632794 GI:1334876 GI:1632787 GI:1632791 GI:1632792

49 16 18 65 66 69 29 29 27 55 15 105

high none medium low very low none low high very low very low medium very low

very low none none very low very low none very low none very low none medium none

accession no.

a Expression level was estimated from Coomassie-stained gels of proteins prepared under denaturing conditions. Expression was designated as “high” if expression was greater than 10 mg/L of insect cells, “medium” if expression was 5 to10 mg/L of insect cells, “low” if expression was 1 to 5 mg/L of insect cells, “very low” if expression was less than 1 mg/L of insect cells but detectable in Coomassie-stained gels or by MALDI-TOF mass spectrometry, and “none” if the protein was not detected by either Coomassie-stained gels or mass spectrometry. b Solubility was estimated from Coomassie-stained gels of proteins prepared under nondenaturing conditions. Amounts of proteins indicated are as defined in a.

baculoviruses were generated for most of the coding sequences, with the exceptions indicated above. The results for each of the 59 HSV-1 proteins, 59 EBV proteins, and 141 CMV proteins

examined are shown in Tables 1, 2 and 3, respectively. In addition, a set of baculoviruses for 78 different vaccinia virus proteins were generated in duplicate and results for each Journal of Proteome Research • Vol. 4, No. 6, 2005 2229

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Table 3. Expression and Solubility of CMV Proteins in Insect Cells expression

ORF/protein name

accession no.

protein size |(kDa with tag)

levela

solubilityb

IRL1 (TRL1) IRL2 (TRL2) IRL3 (TRL3) IRL4 (TRL4) IRL5 (TRL5) IRL6 (TRL6) IRL7 (TRL7) IRL8 (TRL8) IRL9 (TRL9) IRL10 (TRL10) IRL11 (TRL11) IRL12 (TRL12) IRL13 (TRL13) IRL14 J1I TRL14 (RL13) TRS1 UL1 UL2 UL3 UL4 UL5 UL7 UL8 UL9 UL12 UL15 UL17 UL18 UL19 UL20 UL21 UL23 UL24 UL26 UL28 UL29 UL30 UL33 UL35 UL39 UL41 UL43 UL45 UL50 UL51 UL53 UL56 UL58 UL59 UL60 UL62 UL63 UL64 UL65 UL66 UL67 UL68 UL71 UL72 UL73 UL74 UL76 UL77 UL79 UL81 UL82 UL85 UL87 UL91 UL92

gi:124876 gi:124877 gi:124878 gi:137816 gi:124879 gi:124880 gi:124881 gi:124882 gi:124883 gi:124884 gi:124885 gi:124886 gi:124887 gi:124888 gi:125057 gi:9625685 gi:9625873 gi:59606 gi:59607 gi:59608 gi:59609 gi:59610 gi:59612 gi:59613 gi:59614 gi:59618 gi:1780806 gi:59622 gi:59623 gi:59624 gi:59625 gi:59626 gi:1780808 gi:59629 gi:59631 gi:1780809 gi:59634 gi:59635 gi:59638 gi:59640 gi:1780817 gi:1813967 gi:1780821 gi:1780823 gi:1780828 gi:1780829 gi:1780831 gi:1780834 gi:1780836 gi:1780837 gi:1780838 gi:1780840 gi:1780841 gi:1780842 gi:1780843 gi:1780844 gi:1780845 gi:1813969 gi:1780847 gi:1780849 gi:1780850 gi:1780851 gi:1780853 gi:1780854 gi:1780856 gi:1780859 gi:1780860 gi:1780863 gi:1780865 gi:1780868 gi:1780870

37 14 15 22 15 14 12 16 18 21 29 50 18 23 39 24 86 28 9 14 20 21 26 16 29 10 37 15 44 13 41 22 41 42 23 45 43 16 46 75 16 19 23 104 45 19 44 98 16 16 20 26 17 13 14 16 15 15 48 46 17 56 38 73 36 15 64 37 107 14 25

high low very low low low low medium medium medium none none none very low none low none none very low none very low none none none medium none very low very low none none low none very low none high medium very low very low high none high none medium none very low very low medium high very low very low none very low very low none very low none medium very low none low none none none very low low none none very low none none high very low

low low none very low none none very low none very low none none none none none none none none none none none none none none low none very low none none none none none none none none none none none none none medium none medium none none none medium high none none none very low none none none none low none none very low none none none none none none none none none none none none

expression

ORF/protein name

accession no.

protein size |(kDa with tag)

levela

solubilityb

UL93 UL94 UL95 UL96 UL97 UL98 UL99 UL100 UL101 UL103 UL106 UL107 UL108 UL109 UL110 UL111A UL114 UL116 UL119 UL120 UL121 UL122 UL124 UL126 UL127 UL128 UL129 UL130 UL130 (Towne) UL131 UL132 UL133 (Towne) UL134 (Towne) UL137 (Towne) UL140 (Towne) UL141 (Towne) UL142 (Towne) UL144 (Towne) UL145 (Towne) UL148 (Towne) UL150 (Towne) UL151 (Towne) US1 US2 US3 US4 US5 US6 US7 US8 US9 US10 US11 US14 US15 US16 US19 US20 US21 US22 US23 US25 US27 US28 US29 US30 US32 US34 US35 US36

gi:1780871 gi:1780872 gi:1780873 gi:1780875 gi:1780876 gi:1780877 gi:1780878 gi:1780879 gi:1780880 gi:1780882 gi:1780885 gi:1780886 gi:1780887 gi:1780890 gi:1780891 gi:1780892 gi:1780896 gi:1780898 gi:1780901 gi:1780902 gi:1780903 gi:1813972 gi:1780907 gi:1780909 gi:1780910 gi:1780911 gi:1780912 gi:1780913 gi:1167935 gi:1780914 gi:1780915 gi:1167918 gi:1167919 gi:1167922 gi:1167925 gi:1167926 gi:1167927 gi:1167929 gi:1167930 gi:1167933 gi:1167937 gi:1167938 gi:1780932 gi:1780933 gi:1780934 gi:1780935 gi:1780936 gi:1780937 gi:1780938 gi:1780939 gi:1780940 gi:1780941 gi:1780942 gi:1780945 gi:1780946 gi:1780947 gi:1780950 gi:1780951 gi:1780952 gi:1780953 gi:1780954 gi:1780956 gi:1780958 gi:1780959 gi:1780960 gi:1780961 gi:1780963 gi:1780964 gi:1780966 gi:1780967

71 40 59 15 80 67 23 45 14 31 17 19 17 14 16 11 30 40 17 25 22 47 18 18 17 18 15 27 27 10 32 30 22 13 15 51 37 22 13 39 73 38 26 23 24 15 17 23 28 29 30 23 27 36 55 37 29 42 29 69 71 22 44 39 53 41 24 20 15 14

none high very low low none high none none medium very low high none none none none none high none none none none very low none very low low none low none none none none very low low none none medium none none low none high none none very low very low low none none none none none none very low none very low none medium none medium none very low none none very low none none high none low very low

none high none very low none high none none medium none none none none none none none high none none none none none none none very low none very low none none none none none low none none low none none none none none none none none none none none none none none none none none none none none low none none none none none none none none none very low none none none

a Expression level was estimated from Coomassie-stained gels of proteins prepared under denaturing conditions. Expression was designated as “high” if expression was greater than 10 mg/L of insect cells, “medium” if expression was 5 to10 mg/L of insect cells, “low” if expression was 1 to 5 mg/L of insect cells, “very low” if expression was less than 1 mg/L of insect cells but detectable in Coomassie-stained gels or by MALDI-TOF mass spectrometry, and “none” if the protein was not detected by either Coomassie-stained gels or mass spectrometry. b Solubility was estimated from Coomassie-stained gels of proteins prepared under nondenaturing conditions. Amounts of proteins indicated are as defined in a.

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Expression Profiling of Herpesvirus and Vaccinia Virus Table 4. Expression and Solubility of Vaccinia Virus Proteins in Insect Cells ORF/protein name

VACWR012 VACWR016 VACWR017 VACWR018 VACWR021 VACWR022 VACWR024 VACWR026 VACWR028 VACWR029 VACWR030 VACWR034 VACWR035 VACWR036 VACWR038 VACWR040 VACWR042 VACWR043 VACWR044 VACWR046 VACWR054 VACWR056 VACWR057 VACWR059 VACWR060 VACWR063 VACWR069 VACWR090 VACWR091 VACWR093 VACWR094 VACWR096 VACWR099 VACWR105 VACWR106 VACWR107 VACWR112 VACWR114 VACWR119 VACWR120 VACWR125 VACWR127 VACWR131 VACWR135 VACWR138 VACWR139 VACWR143 VACWR145 VACWR146 VACWR147 VACWR149 VACWR161 VACWR164 VACWR166 VACWR167 VACWR169 VACWR171 VACWR173 VACWR174 VACWR175 VACWR177 VACWR179 VACWR180 VACWR188 VACWR192 VACWR193 VACWR194 VACWR195 VACWR196 VACWR198 VACWR202 VACWR203


zinc fingerlike ankyrin-like ankyrin-like C7L, host range C6L C4L C2L, Kelch-like N1L, Virokine N2L, alpha-amanitin target M1L, ankyrin-like, K3L, interferon resistance protein K4L, phospholipase-D-like putative monoglyceride lipase K6L, putative monoglyceride lipase F1L, Protein F1, apoptosis inhibitor F3L, Kelch-like F4L, ribonucleotide reductase small subunit F5L F7L F15L F17R E1L, poly-A polymerase catalytic subunit VP55 E3L, dsRNA binding protein E4L, VITF1, RNA pol subunit rpo30 E7R, myristylprotein O2L, glutaredoxin L3L L4R, core protein vp8 J1R, virion protein J2R,thymidine kinase J4R, RNA pol subunit rpo22 H1L, Tyr/Ser protein phosphatase H7R, late protein H7 D1R, mRNA capping enzyme large subunit D2L, virion core protein D7R, RNA pol subunit rpo18 D9R A1L, VLTF-2 A2L, VLTF-3 A6L A8R, VITF-3 small subunit A12L, core protein A15L, A15 protein A18R, DNA helicase A19L, A19 protein A23R, large subunit of VITF-3 A25L A26L-like A26L-like A39L, semaphorin-like A41L A42R, profilin A45R, superoxide dismutase A47L A48R, thymidylate kinase A49R A51R A53R, secreted TNF-receptor-like, CrmC A55R, Kelch-like B6R, ankyrin-like B10R, Kelch repeat protein B10 B11R B12R, Ser/Thr protein kinase-like B13R, CrmA, IL-1 convertase, Serpin 2 B15R, B15 protein B17L, B17 protein B20R, ankyrin-like Ankyrin repeat protein B19/B20

expression

accession no.


levela

solubilityb

gi:29692118 gi:29692122 gi:29692123 gi:29692124 gi:29692127 gi:29692128 gi:29692130 gi:29692132 gi:29692134 gi:29692135 gi:29692136 gi:29692140 gi:29692141 gi:29692142 gi:29692144 gi:29692146 gi:29692148 gi:29692149 gi:29692150 gi:29692152 gi:29692160 gi:29692162 gi:29692163 gi:29692165 gi:29692166 gi:29692169 gi:29692175 gi:29692196 gi:29692197 gi:29692199 gi:29692200 gi:29692202 gi:29692205 gi:29692211 gi:29692212 gi:29692213 gi:29692218 gi:29692220 gi:29692225 gi:29692226 gi:29692231 gi:29692233 gi:29692237 gi:29692241 gi:29692244 gi:29692245 gi:29692249 gi:29692251 gi:29692252 gi:29692253 gi:29692255 gi:29692267 gi:29692270 gi:29692272 gi:29692273 gi:29692275 gi:29692277 gi:29692279 gi:29692280 gi:29692281 gi:29692283 gi:29692285 gi:29692286 gi:29692294 gi:29692298 gi:29692299 gi:29692300 gi:29692301 gi:29692302 gi:29692304 gi:29692308 gi:29692309

10 11 11 8 20 19 39 61 16 23 56 13 51 7 11 29 58 39 19 10 19 13 58 24 32 22 14 43 31 20 22 23 22 19 99 19 20 27 12 28 45 36 23 13 59 10 47 10 20 29 60 9 18 27 17 11 16 31 28 21 40 14 67 22 21 10 35 41 19 42 8 38

very low none low very low none high none very low medium very low high very low very low none medium very low very low very low low medium very low high medium very low none medium very low very low none very low low medium high low medium low very low very low very low high medium very low very low none very low medium very low very low none none low medium low very low high medium none medium medium medium none high medium low medium high very low none very low medium low none

none none none none none medium none none medium none medium none none none none none none none low none none low medium none none medium very low none none none none very low high low medium low none none none high none none none none very low medium none none none none low none none none high medium none none none medium none none low none medium high none none none medium low none

Journal of Proteome Research • Vol. 4, No. 6, 2005 2231

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Table 4. (Continued) ORF/protein name

expression


accession no.


levela

solubilityb

B21 protein C12L, Serpin 1 ankyrin-like ankyrin-like ankyrin-like

gi:29692310 gi:29692311 gi:29692317 gi:29692319 gi:29692320 gi:29692322

18 43 15 10 8 10

medium none very low medium none none

none none none low none none

VACWR204 VACWR205 VACWR211 VACWR213 VACWR214 VACWR216

a Expression level was estimated from Coomassie-stained gels of proteins prepared under denaturing conditions. Expression was designated as “high” if expression was greater than 10 mg/L of insect cells, “medium” if expression was 5-10 mg/L of insect cells, “low” if expression was 1 to 5 mg/L of insect cells, “very low” if expression was less than 1 mg/L of insect cells but detectable in Coomassie-stained gels or by MALDI-TOF mass spectrometry, and “none” if the protein was not detected by either Coomassie-stained gels or mass spectrometry. b Solubility was estimated from Coomassie-stained gels of proteins prepared under nondenaturing conditions. Amounts of proteins indicated are as defined in a.

Table 5. Summary of Protein Expression and Solubility in Insect Cells for Each Virus virus

no. proteins tested

no. expresseda

no. solubleb

HSV EBV CMV Vaccinia

59 59 141 78

53 (90%) 53 (90%) 71 (50%) 62 (79%)

18 (31%) 13 (22%) 15 (11%) 23 (29%)

a Includes proteins expressed at all levels ranging from very low to high. Includes proteins listed as low to high solubility. Proteins showing very low solubility are not included.

b

protein are shown in Table 4. For each baculovirus, the expression and solubility of the viral protein of the expected size was evaluated under denaturing and nondenaturing conditions, respectively, as described above. The expression level of total protein and soluble protein was estimated from Coomassie-stained gels as high (greater than 10 mg per liter of insect cell culture), medium (5-10 mg per liter), low (1-5 mg per liter) or very low (less than 1 mg per liter). In addition, in cases where no protein was observed in the Coomassiestained gel, samples were analyzed by MALDI-TOF mass spectrometry and classified as “very low” if the target proteins was detected or “none” if it was not. As summarized in Table 5, this baculovirus system successfully produced and purified approximately 90% of the HSV and EBV proteins, 50% of the CMV proteins and 80% of the vaccinia virus proteins, although the amounts of the proteins recovered varied dramatically. In the majority of cases however, most or all of the protein produced was insoluble. In Table 5, we have defined a protein as being soluble if it is recovered under nondenaturing conditions at levels at or above 1 mg/L of culture; proteins classified as “very low” solubility were not included, since they would be difficult to purify in soluble form. A high number of herpesvirus proteins fall into this “very low” solubility category, such that their inclusion increases the number of soluble proteins from 18 to 42 for HSV, from 13 to 38 for EBV and from 15 to 25 for CMV (but little change of 23 to 26 from vaccinia virus). Since all protein expression was tested under a single set of growth conditions, it is possible that the solubility of less soluble proteins may be improved by altering growth conditions, infection time or using a different insect cell line. Comparison to Expression of Viral Proteins in E. coli. We were interested in comparing viral protein expression in insect cells to that in E. coli. While we are aware of several cases where individual proteins express more efficiently in insect cells than in E. coli, a formal comparison of the expression of herpesvirus proteins and vaccinia virus proteins has not to our knowledge 2232


been conducted. Since it is commonly observed that large proteins are particularly difficult to express in E. coli, we selected a set of the smaller (300 amino acids or less) proteins for E. coli expression (32 from HSV, 25 from EBV, 48 from CMV, 81 from vaccinia virus). To select the expression conditions, we initially compared the expression of a set of 27 HSV proteins in BL21 Gold (DE3) cells with and without a plasmid expressing rare-codon tRNAs and found that more proteins were expressed in the presence of the rare-codon tRNAs (data not shown). We then compared the expression of a set of 19 proteins from HSV, EBV, CMV and vaccinia virus in E. coli (with a plasmid expressing rare-codon tRNAs) under two induction conditions (overnight induction at 15 °C and 3 h induction at 37 °C), but found no significant difference in the number of proteins expressed and their solubility in the two induction conditions (data not shown). The 186 viral proteins from the four viruses were expressed with N-terminal hexahistidine tags in small scale cultures in the presence of rare-codon tRNAs and the amount of recombinant protein present in total cell extracts and soluble cell lysates was determined as described in the Experimental Section. The expression level and solubility of individual proteins is presented in Table 6 (in comparison to the same proteins in insect cells) and expression summaries for the set of 152 proteins tested in both E.coli and baculovirus systems are shown for each virus in Table 7. Overall expression of herpesvirus proteins was much lower in E. coli (29%, 28%, and 27% for HSV, EBV, and CMV, respectively) compared to insect cells (90%, 84% and 68% for HSV, EBV, and CMV, respectively). The percentage of proteins expressed in E. coli would be expected to be even lower if larger proteins had been included in the protein set. Most of the individual herpesvirus proteins were expressed at higher levels in insect cells than in E. coli, including 24 that were not expressed at all in E. coli but expressed at medium to high levels in insect cells. Yet, there were a few proteins from each herpesvirus (4 from HSV-1, 2 from EBV and 10 from CMV) that appeared to be expressed at higher levels in E. coli than in insect cells. However, since our baculovirus expression system screens for the purification of the expressed protein (as opposed to expression alone) and our E. coli system screened only for expression level, we do not know whether the apparent increase in expression of these proteins in E. coli is truly due to increased expression levels or reflects the failure of these proteins to bind and elute efficiently from the nickel resin in the baculovirus system. The number of soluble proteins expressed was similar for the two systems, and there was a high degree of overlap in which proteins were soluble. Of the 14 soluble herpesvirus proteins expressed in E.

research articles

Expression Profiling of Herpesvirus and Vaccinia Virus Table 6. Expression and Solubility of Viral Proteins in E. coli as Compared to Insect Cells viral genome

gene name

HSV

ORF O ORF P UL2 UL3 UL4 UL7 UL8.5 UL9.5 UL14 UL15.5 UL18 UL20.5 UL23 UL24 UL27.5 UL31 UL33 UL34 UL35 UL40 UL45 UL49A UL50 UL51 UL55 UL56 US1.5 US2 US8A US9 US11 US12 BALF1 BARF1 BBLF1 BBLF3 BBRF2 BCRF1 BDLF3 BERF1 BFRF2 BFRF3 BILF2 BKRF3 BKRF4 BLLF2 BLLF3 BLRF1 BMRF1 BNLF2a BNLF2b BSRF1 BVLF1.5 BXRF1 BZLF1 BZLF2 EBNA3A IRL4 (TRL4) IRL5 (TRL5) IRL6 (TRL6) IRL7 (TRL7) IRL8 (TRL8) IRL9 (TRL9) IRL14 UL1 UL2 UL7 UL8 UL19 UL21 UL26

EBV

CMV

E. coli expression levela

solubilityb

none none none none none none high none low none high none high none none none none low low high low none low none none none none high none none none none none high none none none none none low none none none high none none high none high none none high none low none none none none none high high high none high none none none low none high high

none none none none none none none none low none medium none high none none none none none none high none none low none none none none low none none none none none none none none none none none low none none none high none none low none medium none none low none none none none none none none none none very low none none none none none low none low low

insect cell expression levelc

low very low high high low medium low high high high high low high very low medium very low medium medium none high none very low high high high very low low low none very low high very low medium none low low very low very low low none medium low high low high high very low high medium medium medium medium low high very low medium low low low medium medium medium none very low none none medium low very low medium

solubilityc

very low very low very low very low none none very low very low low high high none high very low very low very low very low very low none high none none high high medium very low very low very low none none very low very low medium none none none very low none low none none none very low low very low high none high very low none very low none very low none very low medium very low none none very low none very low none none none none low none none none

viral genome

CMV

gene name

UL30 UL43 UL51 UL59 UL60 UL62 UL63 UL64 UL65 UL66 UL67 UL68 UL92 UL96 UL101 UL103 UL107 UL108 UL109 UL110 UL114 UL126 UL128 UL129 UL134 (Towne) UL136 (Towne) UL137 (Towne) UL145 (Towne) UL149 (Towne) US2 US5 US19 US21 US32 Vaccinia VACWR013 VACWR 016 VACWR 017 VACWR 018 VACWR 021 VACWR 027 VACWR 028 VACWR 029 VACWR 031 VACWR 034 VACWR 036 VACWR 037 VACWR 038 VACWR 039 VACWR 040 VACWR 045 VACWR 046 VACWR 047 VACWR 048 VACWR 053 VACWR 054 VACWR 055 VACWR 056 VACWR 059 VACWR 060 VACWR 063 VACWR 067 VACWR 069 VACWR 075 VACWR 079 VACWR 083 VACWR 091 VACWR 094 VACWR 096 VACWR 099 VACWR 105 VACWR 108


solubilityb

none none none high none none none none none none none none none medium none none none none none none none none none none high none none none none none high none none high none none high medium high high high high none medium medium none medium high medium none medium medium medium medium medium medium medium medium none high high none high medium high medium medium high high high medium

none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none low low low none low none none none low none none none none none none low none medium none none none none none none medium none medium none none none low none low low none


solubilityc

high none medium none very low very low none very low none medium very low none very low low medium very low none none none none high very low none low low

none none medium none very low none none none none low none none none very low medium none none none none none high none none very low low

none low

none none

very low none medium medium high

none none low none very low

none low very low none

none none none none

medium very low

medium none

very low none

none none

medium

none

very low

none

medium

none

very low

none

high very low none medium

low none none medium

very low

very low

none low medium high low

none none very low high low


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Gao et al.

Table 6. (Continued) viral genome

gene name

Vaccinia VACWR 109 VACWR 112 VACWR 114 VACWR 115 VACWR 117 VACWR 119 VACWR 120 VACWR 124 VACWR 127 VACWR 131 VACWR 135 VACWR 139 VACWR 140 VACWR 142 VACWR 145 VACWR 146 VACWR 147 VACWR 153 VACWR 154 VACWR 158 VACWR 161 VACWR 164


solubilityb

medium medium medium none low medium medium medium medium high high none high medium high high medium high none high none medium

none none none none none none low none low low high none none none medium low low medium none none none none


solubilityc

very low very low

none none

very low high

none high

very low very low none medium

none none none medium

very low none none

none none none

medium low

none none

viral genome

gene name

Vaccinia VACWR 167 VACWR 171 VACWR 173 VACWR 174 VACWR 175 VACWR 179 VACWR 182 VACWR 185 VACWR 188 VACWR 190 VACWR 191 VACWR 192 VACWR 193 VACWR 194 VACWR 196 VACWR 201 VACWR 202 VACWR 204 VACWR 210 VACWR 211 VACWR 214 VACWR 218


solubilityb

high none high medium medium none high none medium none none medium medium high medium none medium medium none high none high

low none none none none none none none none none none none low none none none low none none none none medium


solubilityc

high none medium medium medium high

high none none none medium none

low

none

medium high very low very low

medium high none none

low medium

low none

very low none

none none

a Expression level was estimated from Coomassie-stained gels of E. coli lysates prepared under denaturing conditions. Expression was designated as “high” if expression was greater than 10 mg/L of E. coli, “medium” if expression was 5-10 mg/L of E. coli, “low” if expression was 1 to 5 mg/L of E. coli, “very low” if expression was less than 1 mg/L of E. coli but detectable in Coomassie-stained gels, and “none” if the protein was not seen in Coomassie-stained gels. b Solubility was estimated from Coomassie-stained gels of E. coli lysates prepared under nondenaturing conditions. Amounts of proteins indicated are as defined in a. c Data are from Tables 1 to 4. Proteins not tested are indicated as “-”.

Table 7. Direct Comparison of Proteins Tested for Expression in Both E. coli and Baculovirus Systems virus

expression system

no. proteins tested

no. expresseda

no. solubleb

HSV HSV EBV EBV CMV CMV Vaccinia Vaccinia

Insect cells E. coli Insect cells E. coli Insect cells E. coli Insect cells E. coli

31 31 25 25 46 46 50 50

28 (90%) 9 (29%) 21 (84%) 7 (28%) 31 (68%) 12 (27%) 40 (80%) 42 (84%)

8 (26%) 6 (19%) 5 (20%) 5 (20%) 7 (16%) 3 (7%) 12 (24%) 18 (36%)

a Includes proteins expressed at all levels ranging from very low to high. Includes proteins listed as low to high solubility. Proteins showing very low solubility are not included.

b

coli, all but 2 were also expressed in the soluble fraction in insect cells. In summary, these results illustrate that the baculovirus system was superior for expression, and at least equal for solubility, for the vast majority of herpesvirus proteins. The expression results for the vaccinia virus proteins in E. coli were considerably different than those of the herpesviruses and quite comparable to protein expression in insect cells, with 80-84% of the tested proteins being expressed in the two systems. There were only 2 proteins that were expressed in insect cells and not expressed in E.coli, and a striking number of proteins (31) were scored as being expressed at higher levels in E. coli than in insect cells, suggesting an advantage of the E. coli system for these proteins. However, as discussed above, we cannot currently distinguish between the possibilities that these proteins are truly expressed at higher levels in E. coli than insect cells or that they just do not purify efficiently on nickel resin. The total number of soluble proteins was similar for E. coli and baculovirus systems, however there was little overlap between proteins which were soluble in the two systems. 2234


Summary A high-throughput baculovirus system is essential for processing large numbers of proteins. We have developed such a system for generating baculoviruses and assessing the expression, solubility and ease of purification of the encoded proteins. This system was extremely successful at producing proteins from all of the viruses examined, although many of the expressed proteins were insoluble. In part, insolubility may reflect the fact that many of the viral proteins are normally found in complexes with other viral proteins. Nonetheless, the ability to generate pure proteins, even under denaturing conditions, will be useful for the generation of antibodies against these proteins. For all three herpesviruses, the baculovirus system was clearly superior for viral protein production as compared to E. coli expression, at least under the set of conditions tested (although solubility was not greatly enhanced). This did not appear to be the case for vaccinia virus, where similar percentages of the proteins were expressed and soluble in both E. coli and baculovirus systems. Two other groups have recently published adaptations to the baculovirus system to facilitate high-throughput screening. Bahia et al.3 studied the feasibility of replacing culture flasks with deep 24-well blocks for early optimization of protein expression prior to large-scale bioreactor experiments. The conclusions drawn on culture volume, cell density, and shaker speed for optimum growth of Sf9 cells are in good agreement with ours. However, we were able to achieve a 2-3-fold increase in protein expression using High-5 cells under similar conditions, which allowed us to purify enough protein to be visualized by Coomassie staining. Chambers et al.4 used a novel plasmid suitable for protein expression in both E. coli and insect cells, as well as a test expression system similar to ours, where High-5 cells were grown in 24-well blocks and protein expression was analyzed under native and denaturing conditions through interactions with Ni-NTA magnetic beads. How-

research articles

Expression Profiling of Herpesvirus and Vaccinia Virus

ever, unlike our system, their method for baculovirus production was not optimized for high-throughput and the viruses used for analysis of small scale protein expression were pretitrated, which is time-consuming. Our system has been adapted for high-throughput screening at each step of bacmid isolation, baculovirus generation and analysis of protein expression, solubility and ease of purification. The high-throughput baculovirus system presented here is amenable to any large scale project and should be useful for the proteomic analyses of cellular genomes.

Acknowledgment. We thank Dr. Karen Mossman for HSV-1 DNA samples and Dr. Jim Smiley, for helpful comments throughout the course of this work. This work was funded by a grant from Genome Canada through the Ontario Genomics

Institute to Affinium Pharmaceuticals and by Affinium Pharmaceuticals. L. F. and G. M. are Canada Research Chairs in Molecular Virology.

References (1) Knipe, D. M.; Howley, P. M. Fields Virology, Fourth ed.; Lippincott Williams and Williams: Philadelphia, 2001. (2) Zhang, R. G.; Skarina, T.; Katz, J. E.; Beasley, S.; Khachatryan, A.; Vyas, S.; Arrowsmith, C. H.; Clarke, S.; Edwards, A.; Joachimiak, A. et al. Structure 2001, 9, 1095-1106. (3) Bahia, D.; Cheung, R.; Buchs, M.; Geisse, S.; Hunt, I. Protein Expr. Purif. 2005, 39, 61-70. (4) Chambers, S. P.; Austen, D. A.; Fulghum, J. R.; Kim, W. M. Protein Expr. Purif. 2004, 36, 40-47.

PR050137U


Expression Profiling of Herpesvirus and Vaccinia Virus Proteins Using

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