Tuning cell cycle of insect cells for enhanced protein production

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Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Tuning cell cycle of insect cells for enhanced protein production

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Hsuan-Chen Wu a,b , Colin G. Hebert a,b , Chi-Wei Hung b,c , David N. Quan a,b , Karen K. Carter b,c , William E. Bentley a,b,c,∗ a

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Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD 20742, USA Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA

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Article history: Received 18 March 2013 Received in revised form 6 August 2013 Accepted 13 August 2013 Available online xxx

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Keywords: Cell cycle cyclin E Eukaryotic cell Recombinant protein RNA interference Baculovirus

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1. Introduction

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The eukaryotic cell cycle consists of many checkpoints during which certain conditions must be met before passing to subsequent stages. These safeguards ensure cells’ integrity and survival, but may also limit growth and protein synthesis in protein production processes. In this work, we employ metabolic engineering principles to “tune” the cell cycle to overcome checkpoint processes in order to facilitate faster cell growth, and independently, arrest the cell cycle in gap1 (G1) phase for greater protein productivity. Specifically, we identified the complete cyclin E (cycE) cDNA sequence from industrially relevant, Trichoplusia ni (T. ni) derived High FiveTM genomes. We then both knocked down (through RNA interference; RNAi) and overexpressed (on an expression plasmid) cycE gene expression to tune the cell phenotype. We successfully up- and down-regulated cycE transcription, enhancing and hindering cell growth, respectively. We also measured the effects of titrated cycE expression on the cell cycle phase distribution. Finally, we investigated the dose-dependent effects of dsCycE on recombinant protein production using the baculovirus expression system and demonstrated a nearly 2-fold increase in expression of model protein (GFPuv). © 2013 Published by Elsevier B.V.

With the advent of whole genome engineering, our ability to metabolically engineer cells has undergone a transformational shift. While many of the first generation applications have been directed at small molecule or commodity-based products from microbial hosts, it will not be long before these tools will be turned toward insect and mammalian genomes for enhancing synthesis of designer proteins (Cobb et al., 2012; Cong et al., 2013; Majors et al., 2009; Sun et al., 2012). Genomewide tools are envisioned to alter global regulatory structures and the overall biosynthetic landscape, while specific bottlenecks are often tackled by transient and local controllers (March and Bentley, 2006, 2007; Ni et al., 2011; Stadlmayr et al., 2010). For example, in many eukaryotic (mammalian, insect, yeast) cell systems, which provide superior glycosylation capabilities,

Abbreviations: cycE, cyclin E; RNAi, ribonucleic acid interference; GFP, green fluorescent protein; CAT, chloramphenicol acetyl transferase; CHO, Chinese hamster ovary; ds, double stranded; G1, gap-1; G2, gap-2; M, mitosis; MOI, multiplicity of infection; S, DNA synthesis; ss, single stranded; T. ni, Trichoplusia ni; RT-PCR, reverse transcription polymerase chain reaction. ∗ Corresponding author at: Fischell Department of Bioengineering, University of Q2 Maryland, College Park, MD 20742. USA. Tel.: +1 301 405 4321; fax: +1 301 405 9953. E-mail address: [email protected] (W.E. Bentley).

interfering RNA (RNAi) has received attention as a means to target local bottlenecks (Hebert et al., 2008; Kim et al., 2012; Lai et al., 2012; March and Bentley, 2007) as well as global processes (Dietzl et al., 2007; Drinnenberg et al., 2009; Xu et al., 2011). Owing largely to superior protein quality, eukaryotic cell cultures remain the main sources for pharmaceutical protein production despite their relatively low growth rates and protein yields as compared to prokaryotic expression systems (Kamionka, 2011; Schmidt, 2004; Terpe, 2006). Low mammalian cell growth rates and yields are due to a complicated network of regulatory mechanisms that monitor and check their internal physiological conditions, metabolic activities, as well as the absence of external signals as a precondition to cell proliferation (Boye and Nordstrom, 2003; Goranov et al., 2009; Slavov and Botstein, 2011; Tapon et al., 2001). Cells progress through chromosome duplication and cell division in a process called the “cell cycle”. The cell cycle consists of many checkpoints during which certain conditions must be met before passing to the next stages. This strategy which ensures cell integrity and survivability can, however, sometimes compromise growth rate and protein synthesis. Since cell cycle and proliferation status are closely related to protein synthesis activity (Gali-Muhtasib and Bakkar, 2002; Kazi and Lang, 2010; Kumar et al., 2007; Stein and Pardee, 2005; Wurm, 2004) the cell cycle has, for many years, been a target of potential metabolic engineering. That is, metabolic

0168-1656/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jbiotec.2013.08.017

Please cite this article in press as: Wu, H.-C., et al., Tuning cell cycle of insect cells for enhanced protein production. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.017

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engineering approaches to arrest the cell cycle at specific states have been successful (Fussenegger et al., 1998; Harper and Brooks, 2005; Sunley and Butler, 2010; Watanabe et al., 2002), but not currently implemented perhaps due to the means of control. For example, chemical signaling additives are typically not desired (Chen et al., 2011; Jiang and Sharfstein, 2008; Sunley and Butler, 2010). The cell cycle can be divided into four distinct phases: gap1(G1), DNA synthesis (S), gap-2 (G2), and mitosis (M) (Cai and Tu, 2012; Harper and Brooks, 2005; van den Heuvel, 2005). Cells arrested at G1 (protein synthesis) phase have been shown to exhibit the highest ribosome biogenesis and protein translation activity of any cell cycle phase, resulting in higher recombinant protein yields (Elledge, 1996; Iba et al., 1978). In G1, cell resources are utilized more efficiently for synthesizing proteins of interests without diverting energy to produce cell components for proliferation (Fussenegger et al., 1998; Mazur et al., 1998; Sunley and Butler, 2010; Watanabe et al., 2002). For example, Bi et al. (2004) showed that over-expression of the cyclin-dependent kinase inhibitor p21 causes G1-phase cell cycle arrest in Chinese hamster ovary (CHO) cells. These G1-arrested cells showed an increase of mitochondrial mass and activity as well as ribosomal protein S6 translation levels, culminating in a 4-fold increase of recombinant protein productivity. Although cell cycle control has been demonstrated in mammalian cell lines for recombinant protein production, few studies have examined the feasibility in insect cell expression systems. March and Bentley (2007) successfully arrested cells in the G1 phase and increased the target GFP production 4-fold in Drosophila S2 cellsusing dsRNA technology (RNA interference) to suppress cyclin E (positive regulator for G1-to-S phase transition) expression. To date, this study is the only reported use of RNAi approaches to control the cell cycle for the purpose of enhancing recombinant protein production in eukaryotic cells. In the current work, we report similar cycE-targeted efforts, but with an industrially relevant insect cell line the High FiveTM cell line derived from the cabbage looper (Trichoplusia ni), the gene for which has previously been unknown. During normal cell proliferation, CycE is regulated so that a natural increase and subsequent decrease guides the G1 to S transition (Cai and Tu, 2012; Stein and Pardee, 2005). Hence, our hypothesis is that altered CycE level can lead to altered cell cycle decision-making and subsequently, altered phenotype. Specifically the sustained upregulation of CycE is hypothesized to yield over-proliferation. Also CycE down-regulation would stall the transition to S from G1. Thus, we investigated the effects of cycE-targeted RNAi molecules that are added to T. ni cells for their impact on regulation of the cell cycle, on cell physiology, and on production of recombinant protein following baculovirus infection. To do this, we identified the complete T. ni cycE cDNA sequence, then up-regulated cycE gene expression by transfecting High FiveTM cultures with an expression vector carrying cycE. Next, cycE gene expression was down-regulated via dsRNA against cycE (denoted dsTnCycE). For both experiments, cycE transcription level, cell growth, as well as the cell cycle distribution (via FACS) were examined. Finally, a dosage-dependent effect of dsTnCycE on recombinant protein production using the baculovirus-mediated expression was revealed. In this study and in our previous work ((Cha et al., 1999b, 1997; Kim et al., 2007; Kramer and Bentley, 2003; Kramer et al., 2003; Wu et al., 2000), GFP is used as a model product in that the results might be transferrable to other more commercially relevant proteins. GFP is a robust protein known to fold properly in a variety of eukaryotic cell lines, hence its use here is reflects a base case for expression without confounding issues relative to posttranslational processing.

2. Materials and methods

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2.1. Cell culture

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T. ni BTI-TN-5B1-4 (High FiveTM , Invitrogen) cells were cultivated in EX-CELLTM -405 media (SAFC Biosciences) at 27 ◦ C in T75 flasks (Corning) for adhesion culture. Cells were also adapted to suspension culture by cultivating in 125 ml shaker flasks (Corning) at ∼120 rpm and at room temperature. Cell passage was conducted every two to three days, or whenever cell density reached confluency (2.5–3 million cells/ml) and then split into 0.6–0.8 million cells/ml. 2.2. Baculovirus amplification and infection A recombinant A. californica multiple nucleopolyhedrovirus (AcMNPV) that expresses GFPuv under the control of the polyhedrin promoter was created previously (Cha et al., 1999a). The baculovirus was amplified in S. frugiperda (Sf9) cells (Invitrogen) according to standard protocols with MOI (=0.1) and supernatants harvested after centrifugation. Baculovirus titer was determined by endpoint dilution with GFPuv fluorescence measurement using fluorescence microscopy (BX60; Olympus), a plate reader (SpectraMax M2; Molecular Devices), or flow cytometry (FACSCanto II; BD Biosciences). 2.3. Identification of T. ni cycE sequence In order to determine the initial putative primers sequences for RT-PCR, cycE sequences among inter-interphylum species were collected from NCBI GenBank and multiple sequence alignment was performed by clustalw2 software (EMBL-EBI) to obtain the consensus regions. Subsequently, degenerate primers for PCR were designed: 5 -GARGARATYTAYCCNCCHAAR-3 (R: A/G; Y: C/T; N: A/T/C/G; H: A/T/C). Next, RACE RT-PCR was performed with the degenerate primers. Briefly, total RNA of High FiveTM cells was extracted using RNAquous (Ambion) followed by reverse-transcription via the SMART-RACETM cDNA amplification kit (Clontech). The resultant PCR products were ligated into a TOPO TA vector (Invitrogen) and subsequent DNA sequencing was carried out by the IBBR sequencing facility (DNA sequencer 3730; Applied Biosystems) to obtain the T. ni cycE cDNA sequence. 2.4. pIB-TncycE vector construction The cycE gene was first PCR amplified using accuprime Hi Fi polymerase (Invitrogen) and TOPO-ligated into a pIB/V5-His vector (Invitrogen) to generate the pIB-TncycE vector. The ligation products were transformed into E. coli TOP 10 competent cells (Invitrogen) and then plated to LB/agar (Fisher Scientific) with Kanamycin 50 ␮g/ml at 37 ◦ C overnight. Single colonies were picked and inoculated into LB broth (Fisher Scientific) with Kanamycin 50 ␮g/ml at 37 ◦ C 250 rpm for overnight. Plasmids were purified with a miniprep kit (Qiagen) and DNA concentrations were quantified by NanoDrop (Thermo Scientific). DNA sequencing of cycE insertion was carried out to confirm cloning success. 2.5. In vitro double stranded RNA (dsRNA) synthesis In vitro doubled stranded CycE (dsTnCycE) RNA was synthesized using the Megascript Kit (Ambion). T7 promoter sequences were incorporated into both the 3 and 5 ends of partial cycE fragments by PCR with primers having extra T7 promoter overhangs. In vitro transcription of dsTnCycE (800 bp) was then generated according to the manufacturer’s instructions. Briefly, single stranded TnCycE was synthesized and then extracted using phenol/chloroform

Please cite this article in press as: Wu, H.-C., et al., Tuning cell cycle of insect cells for enhanced protein production. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.017

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ni mori melanogaster sapiens

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MAQTGS----VCE--------SSEKRLTLKRKRNSTDDELENMPPLKISPVLEEELNDQPAHNVVVESSS MAESGS----PSE--------ASEKRMTLKRKRSSSDDELENQPPQKIASTLDEQLCDQPALHVVVESSS MKLEQKRKFIEMDPELGFEPPSAKRQQRLPALYGSEQGNLSSVASSVYTSPVVSVDGQSTQELLSIRSSP MPRERR----ERD---------AKERDTMK-----EDGGAEFSARSRKRKANVTVFLQDPDEEMAKIDRT

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CSSDDEGSQYGAVDQP------------------------------RSVYTDLDYNPDSFLSPPSISDLP CSSDDEGSQYGNSDQP------------------------------RSVYTDIDYNPDSFLSPPSVSDLP AEDLSEAPHSPLPDSPDSPPSPDRGSKQTPVVVRYAAEQVVTSTVVTQKTEDDDLLDDSCEDYSYDEDDE ARDQCGS----------------------------------------QPWDNNAVCADPCSLIPTPD---

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NCVLSPLENVARGESTPHSNKRPSTSK---------------------------------IPCPTLPKRK NSVLSPLENVARGESTPHSNKRANASK---------------------------------PPCPTPPKRK DDVEEEDDDVEIYSSTISPASSGCSQQQAVNGERTPGLPKHQEQIHHPVSDLMINMRTPMSPAVENGLRQ ----KEDDDRVYPNSTCKPRIIAPS-------------------------------------------RG

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CPLPRLSWADPGDVWNSMCDCDARSSNRKNPNMFDNHPNLQPRMRAILLDWLNEVCEVYKLHRETFHLTV CPLPGLSWADPADVWNSMCECDARSSKKKNPNMFDNHPNLQPRMRAILLDWLNEIGTKKTRQMRVRKLTA CPLPALAWANAADVWRLMCHRDEQDSRLRSISMLEQHPGLQPRMRAILLDWLIEVCEVYKLHRETFYLAV SPLPVLSWANREEVWKIMLNKEK--TYLRDQHFLEQHPLLQPKMRAILLDWLMEVCEVYKLHRETFYLAQ

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DYVDRYLSNTEDVQKGRLQLIGITCLFIAAKVEEVYPPKIGEFAYVTDGACTTDEILLEELLILKILSWS EGFR--------------SQVCITCLFIAAKVEEVYPPKIAEFAYVTDGACTTEEILLEELLILKILSWS DYLDRYLHVAHKVQKTHLQLIGITCLFVAAKVEEIYPPKIGEFAYVTDGACTERDILNHEKILLQALDWD DFFDRYMATQENVVKTLLQLIGISSLFIAAKLEEIYPPKLHQFAYVTDGACSGDEILTMELMIMKALKWR

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ITPITINSWLNVYMQLASEGRSAKRRLLGESDVAANALRSYTFVFPQYSSLEFVICGQLIDLAVLHVDVN ITPITINSWLNVYMQLASEGKSAKRRLLGESDVAANALRGYTFVFPQYSSLEFVICGQLVDLAVLHVDVN ISPITITGWLGVYMQLNVNNRTPAS--FSQIGRQKSAEADDAFIYPQFSGFEFVQTSQLLDLCTLDVGMA LSPLTIVSWLNVYMQVAYLN------------------DLHEVLLPQYPQQIFIQIAELLDLCVLDVDCL

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LFAYSAVAAAAIAHAFTKELAIRVSGYNWDVLEPCWRWLAPFASVIRTEGSVCVVRGGDGEFLQAAGGLD LFSYSAVAAAAIAHTYNRELAMRVSGYKWESLSECYTWLEPFARSLREAGAGGQVRAADGEFVQPAAALR NYSYSVLAAAAISHTFSREMALRCSGLDWQVIQPCARWMEPFFRVISQKAPYLQLNEQN-EQVSNKFGLG EFPYGILAASALYHFSSSELMQKVSGYQWCDIENCVKWMVPFAMVIRETG-------------SSKLKH-

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LICPDVNLDESHRIQSHNVTLDMFDKVYQIMVEQQSVTQTTSEAATSQESEHIYPPTPPAS--------HICPDINPDESHRIQQHNVTLDMFDKVYQAILEHS--SQHDVCPSTSVDTDHIYPPTPPHS--------LICPNIVTDDSHIIQTHTTTMDMYDEVLMAQDAAHAMRARIQASPATALRAPESLLTPPASSHKPDEYLG --FRGVADEDAHNIQTHRDSLDLLDK---ARAKKAMLSEQNRASP-----LPSGLLTPPQS---------

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--------------------------DHKSPKTPTTKTPSTRHLSPPPEARRLSTEaaaaaaaaaaaaaa --------------------------DHKSPKTPTTKTPSNRHEH----ELRIHADaaaaaaaaaaaaaa DEGDETGARSGISSTTTCCNTAASNKGGKSSSNNSVTSCSSRSNP-----------aaaaaaaaaaaaaa --------------------------GKKQSSGPEMA-------------------aaaaaaaaaaaaaa

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Fig. 1. Characterization of T. ni CycE protein sequence (GenBank accession no. JX502019). Amino acid sequence alignment results between T. ni andBombyx mori (silkworm; GenBank accession no. AB457002.2), Drosophila melanogaster (fruit fly; GenBank accession no. NP 476960) and Homo sapiens (human; GenBank accession no. NM 001238). The alignment was performed using ClustalW2 and shading was performed using BOXSHADE. Consensus of at least 50% identical amino acid residues is shaded black; conserved amino acid substitutions are highlighted with gray. Dashes indicate blank positions.

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(Sigma) followed by resuspending in nuclease free water. Subsequently, single stranded TnCycE was re-annealed into dsTnCycE by denaturing at 65 ◦ C for 10 min and then slowly cooling to room temperature. Double stranded chloramphenicol acetyl transferase (dsCAT) was generated the same procedures and served as nonspecific dsRNA for control dsRNA treatment (Hebert et al., 2009).

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RNAi or plasmid vector delivery was carried out utilizing a transfection reagent, Fugene HD (Roche), following manufacturer’s protocols. Varing amounts of RNAi were first diluted into RNase free water (Ambion) followed by mixing with 10 ␮l of Fugene into final 100 ␮l mixtures. The RNAi mixtures were incubated at room temperature for 10 min and then gently added to cell samples. Cells samples were harvested afterward for further analyses.

2.7. Cell cycle analyses For analyzing cell cycle distribution of RNAi – or plasmidtransfected cells, cells were harvested and suspended into 300 ␮l of cold DPBS (Sigma) followed by adding 700 ␮l of 100% cold EtOH dropwise with gentle vortexing, followed by incubation on ice for 15 minutes. Subsequently, cell samples were pelleted and resuspended into a propidium iodide (PI; Sigma) staining solution (50 ␮g/ml PI; 0.1 mg/ml RNase A (Sigma); 0.05% Tritin X-100 in DPBS) for 40 min at 37 ◦ C. The stained samples were pelleted by centrifugation and resuspended back into DPBS for analysis using a FACSCanto II (BD Biosciences) flow cytometer. 2.8. Cell growth Viable cell density of High FiveTM cells was measured using a hemocytometer with trypan blue staining solution to exclude the dead cells.

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To determine relative transcription levels, High FiveTM RNA was extracted (as above), the concentration was quantified by NanoDrop (Thermo Scientific); first strand templates of each sample were synthesized from 500 ng of RNA using a Superscript RT III (Invitrogen) kit with oligo-dT primers. The first strand of cDNA was PCR amplified using cycE primers. Actin primers for PCR amplification were chosen for internal controls of the RT treatments (Hebert et al., 2009). The resultant PCR products were checked by 1% agarose gel electrophoresis (Fisher Scientific) and images were taken with a gel imaging camera (AlphaImager HP; Alpha Innotech).

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3. Results and discussion

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3.1. Identification of T. ni cycE

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We used RACE RT-PCR to identify cycE sequences. Results indicated that the complete cDNA sequence was 1518 bp, which would encode a 506 aa protein (GenBank accession no. JX502019). We performed both nucleotide and protein BLAST analyses, which revealed significant sequence homology with cyclin E families of different eukaryotes. Fig. 1 shows the CycE protein sequence alignment between T. ni and related species, Bombyx mori (silkworm), Drosophila melanogaster (fruit fly), and Homo sapiens (human). Sequence homology was significant but not identical; B. mori had 76.75% identity at the amino acid level; D. melanogaster and H. sapiens were less (both ∼45%). In addition to sequence validation, we used western blot analyses against human cyclin E to further characterize the T. ni CycE at a molecular level. That is, the putative full length cycE was cloned into the insect cell expression vector (pIB/V5-His) yielding plasmid pIB-TncycE, which was transfected into High-fiveTM cells to express a putative histidine-tagged CycE. This protein was histagged purified and probed via rabbit polyclonal anti-human cyclin E. As shown in Supplementary Fig. S1, the western blotting suggested the expressed protein was indeed a CycE. Overall, the newly identified gene and protein product appeared by the physical measurements to belong to family of eukaryotic cyclin E. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2013.08.017 3.2. Manipulating cycE gene expression-effects on cell cycle and cell growth 3.2.1. Up-regulation of cycE gene expression In order to confirm the protein is CycE, we examined its function in T. ni cells. Specifically, the overexpression of CycE in mammalian cells leads to escape from cell cycle regulation and cancerous proliferation (Mishra, 2013; Scaltriti et al., 2011; Vermeulen et al., 2003). Similar phenomena have been reported in insect Drosophila cell cultures and adult flies (Jia et al., 2003; Lai et al., 2005; Simon et al., 2009). This proliferation leads to unchecked progression through G1 to the S phase and a decrease of the G1 population. Here, we characterize transcriptional and phenotypic changes of High FiveTM cells associated with elevated CycE expression associated with transfection with pIB-TncycE. As shown in Fig. 2a (Lane 4), mRNA transcription level increased significantly (∼2 fold) at 3 days post-transfection with pIB-TncycE compared to pIB-egfp, where the transfection efficiency was ∼55–60%, or other controls. In addition, we noted that elevating cycE transcription via pIB-TncycE treatment enhanced cell proliferation (Fig. 2b), particularly as noted 3 days post-transfection. Beyond three days, even for the control cultures, cell densities

Fig. 2. Effects of cycE up-regulation on cell physiology. Initially 0.2 × 106 cells/ml of High FiveTM cells were treated with either pIB-TncycE (for up-regulation), pIBegfp (negative control), or mock transfectant (negative control). Cell samples were harvested at 3 day post transfection and subsequently assayed as follows. (a) mRNA transcription via RT-PCR;(b) cell proliferation via cell count and trypan blue; and (c) cell cycle status via flow cytometry using PI staining.

were close to the maximum supported by the growth medium, suggesting impact of a limiting growth factor. We note that CycE was expressed from the non-replicating plasmids so the effects on regulating networks would be expected to be transient, particularly in rapid growth environments. Finally, while we observed an enhancement in cell growth, no obvious changes of cell cycle distribution were observed (Fig. 2c). It is interesting to note that cell cycle distribution for High-FiveTM cells was found to be heavily skewed toward the G2/M phases (G1: 2.5%, S: 6.5%, G2/M: 65%, >4 N: ∼25%). Not surprisingly, cycE overexpression therefore led to no further distribution shift toward the G2/M phases (Fig. 2c). This result lies in contrast to another industrially relevant insect cell line, Spodoptera frugiperda (Sf9) which under normal cell culture conditions exhibited 29% of the population in G1 (G1: 29%, S: 33%, G2/M: 36%) (Braunagel et al., 1998). Also, we observed a shift in Drosophila S2 cells from 34% to 61% (March and Bentley, 2007). In summary, our results indicate that the High-FiveTM cell growth was influenced by cycE overexpression. 3.2.2. Down-regulation of cycE gene expression In addition to up-regulation, experiments targeting cycE downregulation were conducted. Phenotypic changes to cell cycle phase

Please cite this article in press as: Wu, H.-C., et al., Tuning cell cycle of insect cells for enhanced protein production. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.017

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Multiplicity of Infection Fig. 4. Investigation of dsTnCycE effects on intracellular productivity of adhesion High FiveTM cells using baculovirus expression system. Cells were transfected using various concentrations of dsTnCycE (1 ␮g/ml, 4 ␮g/ml, 8 ␮g/ml, and 12 ␮g/ml) with Fugene for 8 h incubation in prior to infection. Baculovirus AcMNPV that expresses GFPuv under the polyhedrin promoter was utilized for infection at different MOI (0.1, 1, and 10). Cell samples were harvested and fluorescence of intracellular GFPuv was measured by flow cytometry for protein productivity at 4 day post infection.

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Fig. 3. Effects of cycE down-regulation on cell physiology. Suspension High Five cells (0.2 × 106 cells/ml) were treated with dsTnCycE (for silencing), dsCAT (control), and transfectant (Fugene, control). (a) mRNA transcription was performed using RT-PCR at 3 day post transfection. (b) Cell proliferation was performed using cell counting and trypan blue assays. (c) Dosage-dependent cycE silencing on cell cycle was investigated by flow cytometry using PI staining at 3 day post transfection.

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distribution and cell growth were characterized. To down-regulate cycE gene expression, we synthesized dsRNA against T. ni cycE using the method by Hebert et al., 2009. The selected dsTnCycE sequence as amplified was ∼800 bp in length. This relatively long form of dsTnCycE is thought to confer greater stability to dsRNA constructs leading to higher knockdown efficiency of the targeted gene (March and Bentley, 2007). We then performed studies using High-FiveTM suspension cell cultures transfected with either dsTnCycE or a non-specific dsCAT control. The mRNA level of cycE in the experimental population decreased significantly (∼half) compared to the controls (Fig. 3a), suggesting dsTnCycE treatment was successful. Cell growth of CycE silenced populations was nearly abolished (4 ␮g/ml) (Fig. 3b). This reduction of growth rate by knockdown was immediately observed only 1 day after transfection and persisted over the entire time course. During our analysis of the effect of cycE knockdown on cell cycle phase distribution, we discovered a concentration dependent response to dsTnCycE (Fig. 3c). That is, peaks at G2/M phases (2 N) were significantly shifted into G1 (1 N) in a concentration dependent manner, while no shifts were seen for the dsCAT treatment and other controls.

Contrasting this to cycE upregulation suggests that wild type basal CycE abundance in High-FiveTM cells is sufficient for ignoring the G1 to S phase checkpoint – perhaps this feature is one of several molecular characteristics that support its popularity as a host. In summary, functional assays which support over-proliferation in the presence of high cycE expression and cell cycle-arrest in low cycE expression, support our physical characterization results. That is, the sequence depicted in Fig. 1, represents functional full-length T. ni cycE.Enhancing recombinant protein production-effects of CycE RNAi on baculovirus protein expression To investigate altering High-FiveTM cell line productivity through cell cycle modulation, we utilized the baculovirus expression system, one of the most popular insect recombinant protein production platforms. Briefly, High-FiveTM cells adhered to 24-well plates were transfected with a broad range of dsTnCycE concentrations for 8 h, followed by baculovirus (PPH -GFPuv; GFPuv expression under the polyhedrin promoter) infection with various of MOI(0.1, 1, 10) for 4 days. Subsequently, cells were harvested and intracellular GFPuv expression was analyzed by flow cytometry. The resultant fluorescence intensities were further integrated to determine the overall expression level of GFPuv. As depicted in Fig. 4, results displayed enhanced GFP expression up to 2.5fold after treatment with dsTnCycE at high MOI (10). However, at low MOI (0.1 and 1), the effects were not as significant, potentially due to the low expression level of GFPuv. It was also notable that GFPuv expression peaked at 8 ␮g/ml dsTnCycE treatment and dropped significantly at 12 ␮g/ml, potentially indicating the range of dsTnCycE concentration at which cumulative phenotype changes adversely affect overall High-FiveTM cell physiology. Coincidently, similar cycE RNAi toxicity was also observed for Drosophila S2 cell line (∼9 ␮g/ml) for optimized recombinant protein productivity (March and Bentley, 2007). In summary, we demonstrated the use of cycE RNAi to enhance baculovirus driven protein expression in adhered High-FiveTM cells. To elucidate more detail on the effects of CycE RNAi on recombinant protein production, we performed a time series experiment tracking both the intracellular and extracellular GFPuv using High-FiveTM cell suspension culture. Briefly, cell cultures were transfected with various concentrations of dsTnCycE for 8 h, followed by baculovirus infection (MOI = 10) for 2–7 days. In order to establish the definition between intracellular and extracellular GFPuv, 200 ␮l of each cell culture sample was harvested and centrifuged to separate the supernatant (the extracellular

Please cite this article in press as: Wu, H.-C., et al., Tuning cell cycle of insect cells for enhanced protein production. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.08.017

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Fig. 5. Dose-dependent effects of dsTnCycE on intra- and extra-cellular productivity. Cells were transfected with various concentrations of dsTnCycE (2 ␮g/ml, 4 ␮g/ml, 8 ␮g/ml, and 12 ␮g/ml) with Fugene for 8 h incubation in prior to infection. AcMNPV baculovirus producing GFPuv was utilized for infection at MOI 10. The supernatants of the harvested cell samples were collected for extracellular GFPuv measurement. The cell pellets were separately collected and lysed by CytoBusterTM lysis buffer and resulting supernatants were measured as the intracellular GFPuv. Both intra- and extra-cellular GFPuv intensities were read using a plate reader from 2 to 7 day post infection and the fluorescent readings were represented in (a) and (b), respectively. Overall productivity, as shown in (c), was calculated by the summation of the intraand extra-cellular GFPuv intensities.

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increased steadily over time, reaching a zenith at day 5. Comparing the expression at 4 days post infection, the dsTnCycE treated cells were roughly ∼1.8-fold higher than mock infected cells, confirming the integrated FACS results from Fig. 4. Measurements of extracellular GFPuv (Fig. 5b), however, showed a converse outcome where supernatants from control wells had higher GFPuv than treatment wells. Apparently, the dsRNA reduced the release rate of GFPuv from transfected cells. Overall, however, the production of GFPuv (Fig. 5c) derived by combining intracellular and extracellular RFU values, resulted in a nearly 2-fold increase due to the RNAi (at 8 ␮g/ml dsTnCycE). In summary, a cell cycle arrest strategy using dsTnCycE successfully enhanced the cell productivity. We speculate on two potential reasons. First, as mentioned above, dsRNA treatment prolonged the GFPuv production window to the 5th day post infection. This is atypical of baculovirus production where the peak productivity normally appears in 2–4 days. Second, the release of GFPuv into the medium was reduced, which might indicate the baculovirus lytic process was suppressed. This observation had also been hypothesized by Braunagel et al. (1998) who showed that Sf9 cells pre-arrested in G1 and S phases prior to baculovirus infection were unaltered in viral DNA replication, but progeny virus assembly and maturation, e.g. occlusion-derived virus, became abnormal. Perhaps the processes late in the virus life cycle (e.g., polyhedrindriven genes) are most affected by the cell cycle arrest (or visa versa). Further investigations would be necessary to resolve the mechanisms behind these phenomena.

sample) and cell pellet. The cell pellets were then lysed with 200 ␮l CytoBuster TM lysis buffer, centrifuged to pellet cell debris, and the supernatant harvested to produce the intracellular sample. Extracellular and intracellular GFPuv samples were measured by a plate reader and summed to derive a total product yield. As shown in Fig. 5a, the intracellular GFPuv of control samples without dsTnCycE treatment increased significantly by day 3 then dropped thereafter; GFPuv expression of dsTnCycE treated cells, on the contrary,

We have successfully identified the complete cyclin E cDNA sequence of T. ni and characterized recombinant CycE through physical and functional analyses. We have observed that the up-regulation of cycE expression actively facilitated cell growth without perturbing the cell cycle distribution; meanwhile, downregulation of cycE expression through in vitro dsTnCycE caused cell cycle arrest at the G1 phase and inhibited cell growth significantly. Finally, we demonstrated the concentration-dependent dsTnCycE silencing effect on enhancing both the intra- and extra-cellular GFPuv production of T. ni cell culture via the baculovirus expression system. An optimal concentration of RNAi for protein productivity was identified. This RNAi strategy based on altering cell cycle regulation, unlike some procedures using chemicals, is biologically based and is likely transient, which might be advantageous in that it extends cell longevity and productivity while not introducing chemical compounds that might otherwise appear in product formulations. That is, we can envision a stable host cell line that provides transient regulation of cell cycle via an internally triggered RNAi switch, perhaps mediated by the subsequent baculovirus infection. This approach which targets a global condition, using a simple molecular-based switch, holds promise to enhance yield particularly given recent advances in genome engineering of host cells. Acknowledgements The authors greatly appreciate the help from Dr. John C. March at Cornell University for helpful discussions. Partial support of this work was provided from the NSF (EFRI-735987) and the DOD (DTRA #BO08SPO008). References Bi, J.X., Shuttleworth, J., Al-Rubeai, M., 2004. Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol. Bioeng. 85, 741–749.

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