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Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells
GENE-40111; No. of pages: 4; 4C: Gene xxx (2014) xxx–xxx
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Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells Chuanying Pan a,b,⁎,1, Wenchao Jia c,1, Baisong Lu b, Colin E. Bishop b,⁎⁎ a b c
College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, PR China Institute for Regenerative Medicine, Wake Forest University, 391 Technology Way, Winston-Salem, NC 27101, USA College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China
a r t i c l e
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Article history: Received 18 October 2014 Received in revised form 21 November 2014 Accepted 29 November 2014 Available online xxxx Keywords: Recombinant protein Induced pluripotent stem cells (iPSCs) Gene expression Sf9 insect cells
a b s t r a c t Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36 kD, 40 kD, 24 kD, and 36 kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirusinfected Sf9 insect cells will be the method of choice for cellular reprogramming. © 2014 Published by Elsevier B.V.
1. Introduction Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by ectopic expression of defined transcription factors (Takahashi and Yamanaka, 2006). The cells were reprogrammed to resemble embryonic stem cells (ESCs). Owing to their abilities to replicate indefinitely and to differentiate into numerous cell types, generation of iPSCs was thought to be one of the most important breakthroughs in the field of stem cell research (Inoue et al., 2014). Since iPSCs were first generated by Yamanaka et al., many types of cells from different animals and tissues have been successfully induced into the pluripotent state using various methods. However, some problems of cellular reprogramming remain unresolved. The integration of foreign genes into the host genome of virus usage raises a major safety issue, and it has stymied the potential clinical applications of iPSCs. To introduce reprogramming transcription factors into somatic cells while minimizing or avoiding insertion mutagenesis, several techniques that use virus-free systems have been developed. These include the use of non-integrating adenoviruses (Stadtfeld et al., 2008) and Sendai viruses (Ban et al., 2011), plasmids (Okita et al., 2010), PiggyBac
Abbreviations: iPSCs, induced pluripotent stem cells (iPSCs); TAT, transactivator of transcription; BEVS, baculovirus expression vector system ⁎ Correspondence to: C. Pan, College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, PR China. ⁎⁎ Corresponding author. 1 These two authors contributed equally to this work and shared the first authorship.
transposons (Woltjen et al., 2009), episomal vectors (Yu et al., 2009), and small molecule compounds (Hou et al., 2013). Although these methods significantly reduced genome integration, the DNA of the reprogramming factors and vectors remaining after excision, which can also cause insertion mutagenesis; as a consequence, a nucleic acid-free system is highly desirable. To date, virus-gene-free and transgene-free iPSCs have been derived through ectopic expression of transcription factors using reprogramming proteins (Zhou et al., 2009; Pan et al., 2010; Zhang et al., 2012; Nemes et al., 2014) and small molecule compounds (Hou et al., 2013). The most effective method for avoiding DNA integration into the host-cell genome developed to date is to deliver the inducing factors as recombinant proteins. The critical step in this process is the production of large quantities of pure and bioactive proteins. The baculovirus expression vector system (BEVS) and Sf9 insect cells are the ideal choices for recombinant protein production. BEVS is widely used for production of recombinant proteins in insect cells (Mahammad, 2015; Shen et al., 2014), and it was instrumental in heterologous expression of G protein-coupled receptors (GPCRs) and multi-protein complexes for structural studies (Cherezov et al., 2007; Rasmussen et al., 2007; Imasaki et al., 2011; Bieniossek et al., 2012; White et al., 2012). Transient gene expression using BEVS has been well-established for the rapid production of recombinant proteins from mammalian cells, and volumetric yields as high as 1 g/L have been achieved (Backliwal et al., 2008; Geisse, 2009; Rajendra et al., 2011; Hacker et al., 2013). Because c-Myc and Klf4 possess tumorigenicity, the utility of our method was demonstrated to produce a reprogramming cocktail of Oct4, Sox2, Lin28, and Nanog, which are
http://dx.doi.org/10.1016/j.gene.2014.11.061 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
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C. Pan et al. / Gene xxx (2014) xxx–xxx
known as Thomson factors (Yu et al., 2009). All these proteins were fused to the Trans-Activator of Transcription (TAT) cell-penetrating motif. The TAT sequence was derived from the human immunodeficiency virus type 1 (HIV1), and it facilitates cell penetration of the recombinant proteins (Vijaya, 2013). We therefore proposed to test the hypothesis that recombinant reprogramming proteins expressed in Sf9 insect cells, carrying the TAT cell-penetrating motif, would be able to enter somatic cells and reprogram them without involvement of a virus. If this can be achieved, it will be the method of choice for reprogramming in any species, especially humans and domestic animals, that much more valuable as a potential clinical tool for tissue engineering and germplasm resource conservation. 2. Material and methods 2.1. cDNA cloning and plasmids construction The cDNAs of reprogramming inducing factors Oct4, Sox2, Lin28 and Nanog were amplified by PCR, using Pfu DNA polymerase (Clontech, USA). cDNA from hES cells was used as the template to amplify Lin28 and Nanog. A plasmid containing Oct4 (Clone ID: 40125986, Open Biosystems, USA) and a plasmid containing Sox2 (Clone ID: 2823424, Open Biosystems, USA) were used as the template to amplify Oct4 and Sox2, respectively. A 9-amino acid (RKKRRQRRR) membranepenetrating domain (MPD) from the TAT protein of HIV was added at the N-terminus using modified sense primers, shown in Table 1. The PCR products were then sub-cloned into pCR4-TOPO vectors (Invitrogen, USA). After sequence verification, they were restricted with EcoRI/SmaI/SrfI and NotI, followed by sub-cloning into the equivalent sites of the baculovirus transfer plasmid, pBAC-3 (Novagen, Nottingham, UK). The resulting clones were then verified by sequencing, and were referred to herein as pBAC-3/TAT-gene. 2.2. Cell culture Sf9 insect cells were adapted to growth in suspension or monolayer in Sf-900 II SFM (Life Technologies, Basel, Switzerland). Suspension culture cells were usually seeded at 0.5 × 106 cells/mL in a total volume of 50 mL in a 250-mL disposable plastic Erlenmeyer flask. Exponentially growing cells were incubated in a temperature-controlled orbital shaker at 28 °C, 150 rpm. Cells were split when the density reached 4 × 10 6 cells/mL. Typically, cells grown at 28 °C in a monolayer were split 1:8 every 3–4 days, if the cells were healthy and confluent (85–95%). Cell density and viability were determined by the Trypan Blue exclusion method. 2.3. Expression of fusion proteins A BacMagic Transfection Kit (Novagen, Nottingham, UK), including BacMagic DNA, Insect GeneJuice Transfection Reagent, a Transfection Control Plasmid, Sf9 Insect Cells, and BacVector Insect Cell Medium,
was used for expression of fusion proteins, according to the recommended protocol. Instead of a recombinant transfer plasmid, a corresponding amount of medium and 500 ng of the supplied Transfection Control Plasmid were used as negative and positive transfection controls, respectively. After 5 days of incubation, medium containing seed stock of the recombinant baculovirus was harvested. The virus was amplified in a suspension culture at a low multiplicity of infection (MOI), according to the recommended protocol. When cells appeared to be well infected with virus (usually 3–5 days), the cell culture medium was harvested by centrifugation at 1000 ×g for 20 min at 4 °C. The supernatant was removed aseptically and stored (recombinant virus) in dark at 4 °C, or at − 80 °C for long-term preservation. The virus titer was quickly determined by the help of FastPlax™ Titer Kit (Novagen, Nottingham, UK) before the virus was used in subsequent experiments. For expression, expression constructs in the pBAC3/TAT-gene baculovirus transfer plasmid were transfected into Sf9 insect cells, and Sf9 cells were then infected at a high MOI (approx. 10 pfu/cell) of recombinant virus to ensure that all cells were simultaneously infected and the culture was synchronous, according to the recommended protocol. To determine the optimal time for expression of fused proteins after recombinant virus infection, protein expression of TAT-Sox2 was evaluated at different time points after recombinant virus infection (0 h, 48 h, 72 h, 96 h, and 120 h). Insect PopCulture® Reagent with Benzonase® Nuclease or Cell lysis buffer II (10% glycerol, 20 mM HEPES, 1.2 M KCl, 1.5 M MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1% Triton X-100, 0.94 mM spermidine, 15 mM spermine) was then used to harvest cells and cell culture medium, and SDS-PAGE followed by Coomassie Blue staining or Western Blot analysis was used to evaluate protein expression. 2.4. Western Blot analysis Cell lysate was separated using 8–16% SDS-polyacrylamide Ready Gel precast gels (BIO-RAD, USA), electro-transferred to nitrocellulose membranes (Amersham Biosciences, Germany), and blocked with 5% (w/v) dried milk in 1% TBST. The membrane was probed with primary antibody (anti-His probe antibody: Sc-803; Santa Cruz Biotechnology, USA) diluted 1:1000, followed by incubation with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Thermo Fisher Scientific Inc., USA) diluted 1:1000. The bound antibodies were visualized by enhanced chemiluminescence (Western Blotting Luminol Reagent: sc-2048, Santa Cruz Biotechnology, Inc.) and detected by an LAS-3000 imaging system. 2.5. Protein purification Since Oct4 and Sox2 were included in both Yamanaka factors and Thomson factors, which indicated that these two factors were of crucial importance in somatic cell reprogramming. Herein, the TAT-Oct4 and TAT-Sox2 fusion proteins were first purified with Ni-NTA-agarose. Large-scale expression of the recombinant baculovirus-infected Sf9 cells was carried out in 1-L plastic shaker flasks with vented caps
Table 1 Primers used for cloning. Primers
Sequences
GenBank Acc. No.
Lin28-F Lin28-R Nanog-F Nanog-R Oct4-F Oct4-R Sox2-F Sox2-R
5′-GGAATTCCCGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGGCTCCGTGTCCAACCAG-3′ 5′-ATAGCGGCCGCTCAATTCTGTGCCTCCGGGAG-3′ 5′-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGAGTGTGGATCCAGCTTGTC-3′ 5′-ATAGCGGCCGCTCACACGTCTTCAGGTTGCATG-3′ 5′-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGCGGGACACCTGGCTTCG-3′ 5′-ATAGCGGCCGCTCAGTTTGAATGCATGGGAGAGC-3′ 5′-TCGCCCGGGCGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGTACAACATGATGGGAGCG-3′ 5′-ATAGCGGCCGCTCACATGTGTGAGAGGGGCAG-3′
NM_024674 NM_024674 NM_024865 NM_024865 BC117435 BC117435 BC013923 BC013923
Note: The underlines indicated the restriction enzyme cutting sites.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
C. Pan et al. / Gene xxx (2014) xxx–xxx
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(200 mL of culture per flask) in Sf-900 II SFM (Life Technologies, Basel, Switzerland). When Sf9 cells appeared to be well-infected, the cells were lysed with Insect PopCulture® Reagent and Benzonase® Nuclease, according to the recommended protocols. After removal of cell debris by centrifugation, 1 mL of a 50% Ni-NTA slurry was added to 4 mL of lysate and mixed by shaking for 30 min at room temperature. The lysate–resin mixture was carefully loaded into an empty column and washed twice with 4 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Finally, proteins were eluted using an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). 3. Results and discussion 3.1. Identification of recombinant proteins by Coomassie Blue staining and Western Blot analysis To determine the optimal time for fused protein expression, the expression of TAT-Sox2 was evaluated at different time points (0 h, 48 h, 72 h, 96 h, and 120 h) after recombinant virus infection. The result of Coomassie Blue staining indicated that, the target protein was detected only in the cell lysate, and not in the cell culture medium (data not shown). Western Blot analysis further indicated that TAT-Sox2 was detectable in Sf9 cell lysate supernatant and the cell debris after 48 h of virus infection, and the expression was maximal at approximately 72 h post-infection (shown in Fig. 1). In subsequent experiments, we collected the cells at 72 h post-infection. For further identification, the eukaryotic expression recombinant proteins were analyzed by SDS-PAGE followed by Western Blot analysis. As shown in Fig. 2, all four TAT-fused proteins were expressed in the host cells, and all were shown to react with a rabbit polyclonal antibody to the 6xHis probe (shown with arrows). However, each also showed an extra degraded band. This result also demonstrated that the fusion proteins of these four factors were mainly existed in cell lysates, but scarcely detectable as secreted proteins in the cell medium. 3.2. Purification of recombinant TAT-Oct4 and TAT-Sox2 Further study indicated that the fusion proteins were existed in the cell debris but not in the supernatant of the lysate. To extract the TATOct4 and TAT-Sox2 recombinant proteins from the cell nuclei for purification, lysis buffer II was used to lyse the target cells. The supernatant and cell debris were separated and analyzed by SDS-PAGE and Western Blot analysis. As shown in Fig. 3, TAT-Oct4 was only weakly detectable in the supernatant, and more than 95% of this protein was existed in the cell debris. However, TAT-Sox2 could be detected in both the cell lysate supernatant and cell debris, in approximately equivalent amounts. Since the TAT-inducing factor gene was co-expressed with an Nterminal 6xHis tag in Sf9 cells, and Oct4 and Sox2 were the most important reprogramming factors. Here we proposed to purify the TAT-Oct4 and TAT-Sox2 fusion proteins with Ni-NTA-agarose under natural conditions, and then purify the other two proteins as described in the Materials and methods section. We first tried to purify the TAT-Oct4
Fig. 1. Western Blot analysis of TAT-Sox2 expression at different time points after virus infection. Sf9 cells were infected with pBAC-3 recombinant viruses expressing TAT-Sox2 recombinant fusion protein. The supernatant and the deposition of cell lysate were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. The odd number lanes (Lane 1, Lane 3, Lane 5, Lane 7, Lane 9) represented the supernatants of cell lysate, and those cells were infected for 48 h, 72 h, 96 h, and 120 h, respectively. The even number lanes (Lane 2, Lane 4, Lane 6, Lane 8, Lane 10) represented the depositions of cell lysate, and those cells were infected for 48 h, 72 h, 96 h, and 120 h, respectively. Lane M, biotinylated protein ladder.
Fig. 2. Western Blot analysis of 4 recombinant fusion proteins. Sf9 cells were infected with pBAC-3 recombinant viruses expressing 4 recombinant fusion proteins. The supernatant and the deposition of cell lysate were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane M, pre-stained protein ladder; Lanes 1 & 2, the supernatant and deposition of the cell lysate those transfected with pBAC-3/ TAT-Oct4, respectively. Lane 3, Lane 5 and Lane 7, represented the cell culture medium of Sf9 cells transfected with pBAC-3/TAT-Lin28, pBAC-3/TAT-Nanog and pBAC-3/TATSox2, respectively. Lane 4, Lane 6 and Lane 8, represented the cell lysate of Sf9 cells transfected with pBAC-3/TAT-Lin28, pBAC-3/TAT-Nanog and pBAC-3/TAT-Sox2, respectively. Lane 9, bacterial TAT-Sox2 fusion protein. The bigger bands (arrowed) were the TAT-inducing factor target proteins, and the lower bands were the degraded proteins.
recombinant protein, but almost no protein was purified; this result was not entirely unexpected (data not shown). As shown in Fig. 4, more than 80% of the TAT-Sox2 fused protein was existed in the flowthrough. In this study, we aimed to test the feasibility of using BEVS, which is the most widely used approach to the transient production of recombinant proteins from Sf9 cells, for production of recombinant reprogramming proteins. At present, Escherichia coli and mammalian cells have been used to produce defined transcription proteins for generation of iPSCs (Zhou et al., 2009; Kim et al., 2009; Li et al., 2014). The E. coli expression system can produce a high yield of target protein in a short time with low cost, and the protein purification procedure is simple. However, it possesses a major disadvantage, in that the recombinant proteins must be released from the inclusion bodies under denaturing conditions. Since the E. coli expression system is a prokaryotic expression system, the expressed proteins must be solubilized, refolded, and purified. It may be that all of these required treatments impaired the bioactivity of the recombinant proteins, and rendered them useless for reprogramming. BEVS possesses some advantages over the E. coli expression system, compared with other eukaryotic expression systems. First, it can express exogenous genes, particularly those that encode intracellular proteins, at high levels. Second, in most cases, the recombinant proteins are soluble and have been correctly post-translationally modified and folded. Thus, the recombinant proteins possess biological activity, and are easily separated and purified. Third, insect cells can be cultured in suspension. Their easily scalable culture is conducive to large-scale expression of recombinant
Fig. 3. Comparison of the expressions of TAT-Oct4 and TAT-Sox2. Sf9 cells were infected with pBAC-3 recombinant viruses' expressing TAT-Oct4 and TAT-Sox2 recombinant fusion proteins, respectively. Cells were lysed with the cell lysis buffer II to extract the target proteins to the supernatant. The supernatant and the deposition of cell lysate were separated and analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane M, pre-stained protein ladder; Lanes 1 & 2, the supernatant and deposition of cells infected with pBAC-3/TAT-Oct4 virus, respectively; Lanes 3 & 4, the supernatant and deposition of cells infected with pBAC-3/TAT-Sox2 virus, respectively.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
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C. Pan et al. / Gene xxx (2014) xxx–xxx
References
Fig. 4. Purification of TAT-Sox2. For protein expression, 50 mL Sf9 insect suspension culture cells (2 × 106 cells/mL) were infected with recombinant TAT-Sox2 encoding baculovirus at MOI of 10 and 72 h after infection, cells were lysed with Insect PopCulture®Reagent and Benzonase Nuclease, according to the recommended protocols. 1 mL of a 50% Ni-NTA slurry was added to 4 mL clarified lysate, joined together, resulting in a 2.0-mL bed volume for automated liquid chromatography. Crude extract, flow-through, wash and eluate fractions were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane 1, crude extract of infected Sf9 cells; Lane 2, flow-through; Lanes 3–4, first and second washes; Lanes 5–9, eluate fractions.
proteins. And fourth, the most important point is that the baculovirus cannot infect vertebrates (Chen et al., 2012). This characteristic enables the widespread use of BEVS and makes it safe for cellular reprogramming. Recently, a large number of papers have been published that focused on the safety and efficiency of generation of iPSCs, especially safety (Zhou et al., 2009; Kim et al., 2009). Although the recombinant proteininduced method is somewhat inefficient, it is safer than the nuclear transfer method. Here, we intended to reprogram somatic cells using recombinant proteins combined with small molecule compounds. All four fused proteins were expressed in baculovirus-infected Sf9 cells. However, none of these recombinant proteins were expressed as secreted proteins; all of them were detected only in the cell debris. This characteristic of these proteins made purification difficult and limited their utility for cellular reprogramming. These fused proteins may all have been nuclear localized, owing to the presence of nuclear localization signal (NLS) sequences in the transcription factor genes Oct4 and Sox2. If violent methods were used to extract the fused proteins from the cell nuclei, the structure and bioactivity of these proteins might be destroyed, perhaps making them useless for cellular reprogramming. In future, on one hand, we may attempt to use cell extractions of baculovirus-infected Sf9 cells combined with small molecule compounds to reprogram somatic cells. On the other hand, we plan to optimize the expression and purification systems, to produce better results.
Conflict of interests All authors have no conflict of interests.
Acknowledgments This work was supported by the National Natural Science Foundation of Shaanxi Province, China (No. 2014JQ3104), the National Natural Science Foundation of China (No. 31000655) and the National Institutes of Health (NIH) (R21 RR025408).
Backliwal, G., Hildinger, M., Hasija, V., Wurm, F.M., 2008. High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol. Bioeng. 99, 721–727. Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S., 2011. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc. Natl. Acad. Sci. 108, 14234–14239. Bieniossek, C., Imasaki, T., Takagi, Y., Berger, I., 2012. MultiBac: expanding the research toolbox for multi protein complexes. Trends Biochem. Sci. 37, 49–57. Chen, Y.J., Liu, B.R., Dai, Y.H., Lee, C.Y., Chan, M.H., Chen, H.H., Chiang, H.J., Lee, H.J., 2012. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 493, 201–210. Cherezov, V., Rosenbaum, D.M., Hanson, M.A., Rasmussen, S.G., Thian, F.S., Kobilka, T.S., Choi, H.J., Kuhn, P., Weis, W.I., Kobilka, B.K., Stevens, R.C., 2007. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258–1265. Geisse, S., 2009. Reflections on more than 10 years of TGE approaches. Protein Expr. Purif. 64, 99–107. Hacker, D.L., Kiseljak, D., Rajendra, Y., Thurnheer, S., Baldi, L., Wurm, F.M., 2013. Polyethyleneimine-based transient gene expression processes for suspensionadapted HEK-293E and CHO-DG44 cells. Protein Expr. Purif. 92 (1), 67–76. Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J., Li, H., Zhao, T., Ye, J., Yang, W., Liu, K., Ge, J., Xu, J., Zhang, Q., Zhao, Y., Deng, Hongkui, 2013. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341, 651–654. Imasaki, T., Calero, G., Cai, G., Tsai, K.L., Yamada, K., Cardelli, F., Erdjument-Bromage, H., Tempst, P., Berger, I., Kornberg, G.L., Asturias, F.J., Kornberg, R.D., Takagi, Y., 2011. Architecture of the mediator head module. Nature 475, 240–243. Inoue, H., Nagata, N., Kurokawa, H., Yamanaka, S., 2014. iPS cells: a game changer for future medicine. EMBO J. 33, 409–417. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., Kim, K.S., 2009. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476. Li, Xia, Zhang, Pengfei, Wei, Chao, Zhang, Yunhai, 2014. Generation of pluripotent stem cells via protein transduction. Int. J. Dev. Biol. 58, 21–27. Mahammad, A., 2015. Production and purification of recombinant enteropeptidase expressed in an insect–baculovirus cell system. Prep. Biochem. Biotechnol. 45, 268–278. Nemes, C., Varga, E., Polgar, Z., Klincumhom, N., Pirity, M.K., Dinnyes, A., 2014. Generation of mouse induced pluripotent stem cells by protein transduction. Tissue Eng. Part C Methods 20, 383–392. Okita, K., Hong, H., Takahashi, K., Yamanaka, S., 2010. Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5, 418–428. Pan, C., Lu, B., Chen, H., Bishop, C.E., 2010. Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Mol. Biol. Rep. 37, 2117–2124. Rajendra, Y., Kiseljak, D., Baldi, L., Hacker, D.L., Wurm, F.M., 2011. A simple high-yielding process for transient gene expression in CHO cells. J. Biotechnol. 153, 22–26. Rasmussen, S.G.F., Choi, H.J., Rosenbaum, D.M., Kobilka, T.S., Thian, F.S., Edwards, P.C., Burghammer, M., Ratnala, V.R.P., Sanishvili, R., Fischetti, R.F., Schertler, G.F.X., Weis, W.I., Kobilka, B.K., 2007. Crystal structure of the human beta2 adrenergic G-proteincoupled receptor. Nature 450, 383–387. Shen, X., Sanishvili, R., Hacker, D.L., Baldi, L., Wurm, F.M., 2014. Virus-free transient protein production in Sf9 cells. J. Biotechnol. 171, 61–70. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., Hochedlinger, K., 2008. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Vijaya, Gopal, 2013. Bioinspired peptides as versatile nucleic acid delivery platforms. J. Control. Release 167, 323–332. White, J.F., Noinaj, N., Shibata, Y., Love, J., Kloss, B., Xu, F., Gvozdenovic-Jeremic, J., Shah, P., Shiloach, J., Tate, C.G., Grisshammer, R., 2012. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513. Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P.T., Gertsenstein, M., 2009. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770. Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I.I., Thomson, J.A., 2009. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801. Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J., Jin, Y., 2012. Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials 33, 5047–5055. Zhou, H.Y., Wu, S.L., Joo, J.Y., Zhu, S.Y., Han, D.W., Lin, T.X., Trauger, S., Bien, G., Yao, S., Zhu, Y., 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells Chuanying Pan a,b,⁎,1, Wenchao Jia c,1, Baisong Lu b, Colin E. Bishop b,⁎⁎ a b c
College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, PR China Institute for Regenerative Medicine, Wake Forest University, 391 Technology Way, Winston-Salem, NC 27101, USA College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China
a r t i c l e
i n f o
Article history: Received 18 October 2014 Received in revised form 21 November 2014 Accepted 29 November 2014 Available online xxxx Keywords: Recombinant protein Induced pluripotent stem cells (iPSCs) Gene expression Sf9 insect cells
a b s t r a c t Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36 kD, 40 kD, 24 kD, and 36 kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirusinfected Sf9 insect cells will be the method of choice for cellular reprogramming. © 2014 Published by Elsevier B.V.
1. Introduction Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by ectopic expression of defined transcription factors (Takahashi and Yamanaka, 2006). The cells were reprogrammed to resemble embryonic stem cells (ESCs). Owing to their abilities to replicate indefinitely and to differentiate into numerous cell types, generation of iPSCs was thought to be one of the most important breakthroughs in the field of stem cell research (Inoue et al., 2014). Since iPSCs were first generated by Yamanaka et al., many types of cells from different animals and tissues have been successfully induced into the pluripotent state using various methods. However, some problems of cellular reprogramming remain unresolved. The integration of foreign genes into the host genome of virus usage raises a major safety issue, and it has stymied the potential clinical applications of iPSCs. To introduce reprogramming transcription factors into somatic cells while minimizing or avoiding insertion mutagenesis, several techniques that use virus-free systems have been developed. These include the use of non-integrating adenoviruses (Stadtfeld et al., 2008) and Sendai viruses (Ban et al., 2011), plasmids (Okita et al., 2010), PiggyBac
Abbreviations: iPSCs, induced pluripotent stem cells (iPSCs); TAT, transactivator of transcription; BEVS, baculovirus expression vector system ⁎ Correspondence to: C. Pan, College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, PR China. ⁎⁎ Corresponding author. 1 These two authors contributed equally to this work and shared the first authorship.
transposons (Woltjen et al., 2009), episomal vectors (Yu et al., 2009), and small molecule compounds (Hou et al., 2013). Although these methods significantly reduced genome integration, the DNA of the reprogramming factors and vectors remaining after excision, which can also cause insertion mutagenesis; as a consequence, a nucleic acid-free system is highly desirable. To date, virus-gene-free and transgene-free iPSCs have been derived through ectopic expression of transcription factors using reprogramming proteins (Zhou et al., 2009; Pan et al., 2010; Zhang et al., 2012; Nemes et al., 2014) and small molecule compounds (Hou et al., 2013). The most effective method for avoiding DNA integration into the host-cell genome developed to date is to deliver the inducing factors as recombinant proteins. The critical step in this process is the production of large quantities of pure and bioactive proteins. The baculovirus expression vector system (BEVS) and Sf9 insect cells are the ideal choices for recombinant protein production. BEVS is widely used for production of recombinant proteins in insect cells (Mahammad, 2015; Shen et al., 2014), and it was instrumental in heterologous expression of G protein-coupled receptors (GPCRs) and multi-protein complexes for structural studies (Cherezov et al., 2007; Rasmussen et al., 2007; Imasaki et al., 2011; Bieniossek et al., 2012; White et al., 2012). Transient gene expression using BEVS has been well-established for the rapid production of recombinant proteins from mammalian cells, and volumetric yields as high as 1 g/L have been achieved (Backliwal et al., 2008; Geisse, 2009; Rajendra et al., 2011; Hacker et al., 2013). Because c-Myc and Klf4 possess tumorigenicity, the utility of our method was demonstrated to produce a reprogramming cocktail of Oct4, Sox2, Lin28, and Nanog, which are
http://dx.doi.org/10.1016/j.gene.2014.11.061 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
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C. Pan et al. / Gene xxx (2014) xxx–xxx
known as Thomson factors (Yu et al., 2009). All these proteins were fused to the Trans-Activator of Transcription (TAT) cell-penetrating motif. The TAT sequence was derived from the human immunodeficiency virus type 1 (HIV1), and it facilitates cell penetration of the recombinant proteins (Vijaya, 2013). We therefore proposed to test the hypothesis that recombinant reprogramming proteins expressed in Sf9 insect cells, carrying the TAT cell-penetrating motif, would be able to enter somatic cells and reprogram them without involvement of a virus. If this can be achieved, it will be the method of choice for reprogramming in any species, especially humans and domestic animals, that much more valuable as a potential clinical tool for tissue engineering and germplasm resource conservation. 2. Material and methods 2.1. cDNA cloning and plasmids construction The cDNAs of reprogramming inducing factors Oct4, Sox2, Lin28 and Nanog were amplified by PCR, using Pfu DNA polymerase (Clontech, USA). cDNA from hES cells was used as the template to amplify Lin28 and Nanog. A plasmid containing Oct4 (Clone ID: 40125986, Open Biosystems, USA) and a plasmid containing Sox2 (Clone ID: 2823424, Open Biosystems, USA) were used as the template to amplify Oct4 and Sox2, respectively. A 9-amino acid (RKKRRQRRR) membranepenetrating domain (MPD) from the TAT protein of HIV was added at the N-terminus using modified sense primers, shown in Table 1. The PCR products were then sub-cloned into pCR4-TOPO vectors (Invitrogen, USA). After sequence verification, they were restricted with EcoRI/SmaI/SrfI and NotI, followed by sub-cloning into the equivalent sites of the baculovirus transfer plasmid, pBAC-3 (Novagen, Nottingham, UK). The resulting clones were then verified by sequencing, and were referred to herein as pBAC-3/TAT-gene. 2.2. Cell culture Sf9 insect cells were adapted to growth in suspension or monolayer in Sf-900 II SFM (Life Technologies, Basel, Switzerland). Suspension culture cells were usually seeded at 0.5 × 106 cells/mL in a total volume of 50 mL in a 250-mL disposable plastic Erlenmeyer flask. Exponentially growing cells were incubated in a temperature-controlled orbital shaker at 28 °C, 150 rpm. Cells were split when the density reached 4 × 10 6 cells/mL. Typically, cells grown at 28 °C in a monolayer were split 1:8 every 3–4 days, if the cells were healthy and confluent (85–95%). Cell density and viability were determined by the Trypan Blue exclusion method. 2.3. Expression of fusion proteins A BacMagic Transfection Kit (Novagen, Nottingham, UK), including BacMagic DNA, Insect GeneJuice Transfection Reagent, a Transfection Control Plasmid, Sf9 Insect Cells, and BacVector Insect Cell Medium,
was used for expression of fusion proteins, according to the recommended protocol. Instead of a recombinant transfer plasmid, a corresponding amount of medium and 500 ng of the supplied Transfection Control Plasmid were used as negative and positive transfection controls, respectively. After 5 days of incubation, medium containing seed stock of the recombinant baculovirus was harvested. The virus was amplified in a suspension culture at a low multiplicity of infection (MOI), according to the recommended protocol. When cells appeared to be well infected with virus (usually 3–5 days), the cell culture medium was harvested by centrifugation at 1000 ×g for 20 min at 4 °C. The supernatant was removed aseptically and stored (recombinant virus) in dark at 4 °C, or at − 80 °C for long-term preservation. The virus titer was quickly determined by the help of FastPlax™ Titer Kit (Novagen, Nottingham, UK) before the virus was used in subsequent experiments. For expression, expression constructs in the pBAC3/TAT-gene baculovirus transfer plasmid were transfected into Sf9 insect cells, and Sf9 cells were then infected at a high MOI (approx. 10 pfu/cell) of recombinant virus to ensure that all cells were simultaneously infected and the culture was synchronous, according to the recommended protocol. To determine the optimal time for expression of fused proteins after recombinant virus infection, protein expression of TAT-Sox2 was evaluated at different time points after recombinant virus infection (0 h, 48 h, 72 h, 96 h, and 120 h). Insect PopCulture® Reagent with Benzonase® Nuclease or Cell lysis buffer II (10% glycerol, 20 mM HEPES, 1.2 M KCl, 1.5 M MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1% Triton X-100, 0.94 mM spermidine, 15 mM spermine) was then used to harvest cells and cell culture medium, and SDS-PAGE followed by Coomassie Blue staining or Western Blot analysis was used to evaluate protein expression. 2.4. Western Blot analysis Cell lysate was separated using 8–16% SDS-polyacrylamide Ready Gel precast gels (BIO-RAD, USA), electro-transferred to nitrocellulose membranes (Amersham Biosciences, Germany), and blocked with 5% (w/v) dried milk in 1% TBST. The membrane was probed with primary antibody (anti-His probe antibody: Sc-803; Santa Cruz Biotechnology, USA) diluted 1:1000, followed by incubation with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Thermo Fisher Scientific Inc., USA) diluted 1:1000. The bound antibodies were visualized by enhanced chemiluminescence (Western Blotting Luminol Reagent: sc-2048, Santa Cruz Biotechnology, Inc.) and detected by an LAS-3000 imaging system. 2.5. Protein purification Since Oct4 and Sox2 were included in both Yamanaka factors and Thomson factors, which indicated that these two factors were of crucial importance in somatic cell reprogramming. Herein, the TAT-Oct4 and TAT-Sox2 fusion proteins were first purified with Ni-NTA-agarose. Large-scale expression of the recombinant baculovirus-infected Sf9 cells was carried out in 1-L plastic shaker flasks with vented caps
Table 1 Primers used for cloning. Primers
Sequences
GenBank Acc. No.
Lin28-F Lin28-R Nanog-F Nanog-R Oct4-F Oct4-R Sox2-F Sox2-R
5′-GGAATTCCCGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGGCTCCGTGTCCAACCAG-3′ 5′-ATAGCGGCCGCTCAATTCTGTGCCTCCGGGAG-3′ 5′-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGAGTGTGGATCCAGCTTGTC-3′ 5′-ATAGCGGCCGCTCACACGTCTTCAGGTTGCATG-3′ 5′-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGCGGGACACCTGGCTTCG-3′ 5′-ATAGCGGCCGCTCAGTTTGAATGCATGGGAGAGC-3′ 5′-TCGCCCGGGCGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGTACAACATGATGGGAGCG-3′ 5′-ATAGCGGCCGCTCACATGTGTGAGAGGGGCAG-3′
NM_024674 NM_024674 NM_024865 NM_024865 BC117435 BC117435 BC013923 BC013923
Note: The underlines indicated the restriction enzyme cutting sites.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
C. Pan et al. / Gene xxx (2014) xxx–xxx
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(200 mL of culture per flask) in Sf-900 II SFM (Life Technologies, Basel, Switzerland). When Sf9 cells appeared to be well-infected, the cells were lysed with Insect PopCulture® Reagent and Benzonase® Nuclease, according to the recommended protocols. After removal of cell debris by centrifugation, 1 mL of a 50% Ni-NTA slurry was added to 4 mL of lysate and mixed by shaking for 30 min at room temperature. The lysate–resin mixture was carefully loaded into an empty column and washed twice with 4 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Finally, proteins were eluted using an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). 3. Results and discussion 3.1. Identification of recombinant proteins by Coomassie Blue staining and Western Blot analysis To determine the optimal time for fused protein expression, the expression of TAT-Sox2 was evaluated at different time points (0 h, 48 h, 72 h, 96 h, and 120 h) after recombinant virus infection. The result of Coomassie Blue staining indicated that, the target protein was detected only in the cell lysate, and not in the cell culture medium (data not shown). Western Blot analysis further indicated that TAT-Sox2 was detectable in Sf9 cell lysate supernatant and the cell debris after 48 h of virus infection, and the expression was maximal at approximately 72 h post-infection (shown in Fig. 1). In subsequent experiments, we collected the cells at 72 h post-infection. For further identification, the eukaryotic expression recombinant proteins were analyzed by SDS-PAGE followed by Western Blot analysis. As shown in Fig. 2, all four TAT-fused proteins were expressed in the host cells, and all were shown to react with a rabbit polyclonal antibody to the 6xHis probe (shown with arrows). However, each also showed an extra degraded band. This result also demonstrated that the fusion proteins of these four factors were mainly existed in cell lysates, but scarcely detectable as secreted proteins in the cell medium. 3.2. Purification of recombinant TAT-Oct4 and TAT-Sox2 Further study indicated that the fusion proteins were existed in the cell debris but not in the supernatant of the lysate. To extract the TATOct4 and TAT-Sox2 recombinant proteins from the cell nuclei for purification, lysis buffer II was used to lyse the target cells. The supernatant and cell debris were separated and analyzed by SDS-PAGE and Western Blot analysis. As shown in Fig. 3, TAT-Oct4 was only weakly detectable in the supernatant, and more than 95% of this protein was existed in the cell debris. However, TAT-Sox2 could be detected in both the cell lysate supernatant and cell debris, in approximately equivalent amounts. Since the TAT-inducing factor gene was co-expressed with an Nterminal 6xHis tag in Sf9 cells, and Oct4 and Sox2 were the most important reprogramming factors. Here we proposed to purify the TAT-Oct4 and TAT-Sox2 fusion proteins with Ni-NTA-agarose under natural conditions, and then purify the other two proteins as described in the Materials and methods section. We first tried to purify the TAT-Oct4
Fig. 1. Western Blot analysis of TAT-Sox2 expression at different time points after virus infection. Sf9 cells were infected with pBAC-3 recombinant viruses expressing TAT-Sox2 recombinant fusion protein. The supernatant and the deposition of cell lysate were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. The odd number lanes (Lane 1, Lane 3, Lane 5, Lane 7, Lane 9) represented the supernatants of cell lysate, and those cells were infected for 48 h, 72 h, 96 h, and 120 h, respectively. The even number lanes (Lane 2, Lane 4, Lane 6, Lane 8, Lane 10) represented the depositions of cell lysate, and those cells were infected for 48 h, 72 h, 96 h, and 120 h, respectively. Lane M, biotinylated protein ladder.
Fig. 2. Western Blot analysis of 4 recombinant fusion proteins. Sf9 cells were infected with pBAC-3 recombinant viruses expressing 4 recombinant fusion proteins. The supernatant and the deposition of cell lysate were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane M, pre-stained protein ladder; Lanes 1 & 2, the supernatant and deposition of the cell lysate those transfected with pBAC-3/ TAT-Oct4, respectively. Lane 3, Lane 5 and Lane 7, represented the cell culture medium of Sf9 cells transfected with pBAC-3/TAT-Lin28, pBAC-3/TAT-Nanog and pBAC-3/TATSox2, respectively. Lane 4, Lane 6 and Lane 8, represented the cell lysate of Sf9 cells transfected with pBAC-3/TAT-Lin28, pBAC-3/TAT-Nanog and pBAC-3/TAT-Sox2, respectively. Lane 9, bacterial TAT-Sox2 fusion protein. The bigger bands (arrowed) were the TAT-inducing factor target proteins, and the lower bands were the degraded proteins.
recombinant protein, but almost no protein was purified; this result was not entirely unexpected (data not shown). As shown in Fig. 4, more than 80% of the TAT-Sox2 fused protein was existed in the flowthrough. In this study, we aimed to test the feasibility of using BEVS, which is the most widely used approach to the transient production of recombinant proteins from Sf9 cells, for production of recombinant reprogramming proteins. At present, Escherichia coli and mammalian cells have been used to produce defined transcription proteins for generation of iPSCs (Zhou et al., 2009; Kim et al., 2009; Li et al., 2014). The E. coli expression system can produce a high yield of target protein in a short time with low cost, and the protein purification procedure is simple. However, it possesses a major disadvantage, in that the recombinant proteins must be released from the inclusion bodies under denaturing conditions. Since the E. coli expression system is a prokaryotic expression system, the expressed proteins must be solubilized, refolded, and purified. It may be that all of these required treatments impaired the bioactivity of the recombinant proteins, and rendered them useless for reprogramming. BEVS possesses some advantages over the E. coli expression system, compared with other eukaryotic expression systems. First, it can express exogenous genes, particularly those that encode intracellular proteins, at high levels. Second, in most cases, the recombinant proteins are soluble and have been correctly post-translationally modified and folded. Thus, the recombinant proteins possess biological activity, and are easily separated and purified. Third, insect cells can be cultured in suspension. Their easily scalable culture is conducive to large-scale expression of recombinant
Fig. 3. Comparison of the expressions of TAT-Oct4 and TAT-Sox2. Sf9 cells were infected with pBAC-3 recombinant viruses' expressing TAT-Oct4 and TAT-Sox2 recombinant fusion proteins, respectively. Cells were lysed with the cell lysis buffer II to extract the target proteins to the supernatant. The supernatant and the deposition of cell lysate were separated and analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane M, pre-stained protein ladder; Lanes 1 & 2, the supernatant and deposition of cells infected with pBAC-3/TAT-Oct4 virus, respectively; Lanes 3 & 4, the supernatant and deposition of cells infected with pBAC-3/TAT-Sox2 virus, respectively.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061
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References
Fig. 4. Purification of TAT-Sox2. For protein expression, 50 mL Sf9 insect suspension culture cells (2 × 106 cells/mL) were infected with recombinant TAT-Sox2 encoding baculovirus at MOI of 10 and 72 h after infection, cells were lysed with Insect PopCulture®Reagent and Benzonase Nuclease, according to the recommended protocols. 1 mL of a 50% Ni-NTA slurry was added to 4 mL clarified lysate, joined together, resulting in a 2.0-mL bed volume for automated liquid chromatography. Crude extract, flow-through, wash and eluate fractions were analyzed by SDS-PAGE and Western Blot. Primary antibody: His Probe (H-15) antibody, sc-803. Lane 1, crude extract of infected Sf9 cells; Lane 2, flow-through; Lanes 3–4, first and second washes; Lanes 5–9, eluate fractions.
proteins. And fourth, the most important point is that the baculovirus cannot infect vertebrates (Chen et al., 2012). This characteristic enables the widespread use of BEVS and makes it safe for cellular reprogramming. Recently, a large number of papers have been published that focused on the safety and efficiency of generation of iPSCs, especially safety (Zhou et al., 2009; Kim et al., 2009). Although the recombinant proteininduced method is somewhat inefficient, it is safer than the nuclear transfer method. Here, we intended to reprogram somatic cells using recombinant proteins combined with small molecule compounds. All four fused proteins were expressed in baculovirus-infected Sf9 cells. However, none of these recombinant proteins were expressed as secreted proteins; all of them were detected only in the cell debris. This characteristic of these proteins made purification difficult and limited their utility for cellular reprogramming. These fused proteins may all have been nuclear localized, owing to the presence of nuclear localization signal (NLS) sequences in the transcription factor genes Oct4 and Sox2. If violent methods were used to extract the fused proteins from the cell nuclei, the structure and bioactivity of these proteins might be destroyed, perhaps making them useless for cellular reprogramming. In future, on one hand, we may attempt to use cell extractions of baculovirus-infected Sf9 cells combined with small molecule compounds to reprogram somatic cells. On the other hand, we plan to optimize the expression and purification systems, to produce better results.
Conflict of interests All authors have no conflict of interests.
Acknowledgments This work was supported by the National Natural Science Foundation of Shaanxi Province, China (No. 2014JQ3104), the National Natural Science Foundation of China (No. 31000655) and the National Institutes of Health (NIH) (R21 RR025408).
Backliwal, G., Hildinger, M., Hasija, V., Wurm, F.M., 2008. High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol. Bioeng. 99, 721–727. Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S., 2011. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc. Natl. Acad. Sci. 108, 14234–14239. Bieniossek, C., Imasaki, T., Takagi, Y., Berger, I., 2012. MultiBac: expanding the research toolbox for multi protein complexes. Trends Biochem. Sci. 37, 49–57. Chen, Y.J., Liu, B.R., Dai, Y.H., Lee, C.Y., Chan, M.H., Chen, H.H., Chiang, H.J., Lee, H.J., 2012. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 493, 201–210. Cherezov, V., Rosenbaum, D.M., Hanson, M.A., Rasmussen, S.G., Thian, F.S., Kobilka, T.S., Choi, H.J., Kuhn, P., Weis, W.I., Kobilka, B.K., Stevens, R.C., 2007. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258–1265. Geisse, S., 2009. Reflections on more than 10 years of TGE approaches. Protein Expr. Purif. 64, 99–107. Hacker, D.L., Kiseljak, D., Rajendra, Y., Thurnheer, S., Baldi, L., Wurm, F.M., 2013. Polyethyleneimine-based transient gene expression processes for suspensionadapted HEK-293E and CHO-DG44 cells. Protein Expr. Purif. 92 (1), 67–76. Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J., Li, H., Zhao, T., Ye, J., Yang, W., Liu, K., Ge, J., Xu, J., Zhang, Q., Zhao, Y., Deng, Hongkui, 2013. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341, 651–654. Imasaki, T., Calero, G., Cai, G., Tsai, K.L., Yamada, K., Cardelli, F., Erdjument-Bromage, H., Tempst, P., Berger, I., Kornberg, G.L., Asturias, F.J., Kornberg, R.D., Takagi, Y., 2011. Architecture of the mediator head module. Nature 475, 240–243. Inoue, H., Nagata, N., Kurokawa, H., Yamanaka, S., 2014. iPS cells: a game changer for future medicine. EMBO J. 33, 409–417. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., Kim, K.S., 2009. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476. Li, Xia, Zhang, Pengfei, Wei, Chao, Zhang, Yunhai, 2014. Generation of pluripotent stem cells via protein transduction. Int. J. Dev. Biol. 58, 21–27. Mahammad, A., 2015. Production and purification of recombinant enteropeptidase expressed in an insect–baculovirus cell system. Prep. Biochem. Biotechnol. 45, 268–278. Nemes, C., Varga, E., Polgar, Z., Klincumhom, N., Pirity, M.K., Dinnyes, A., 2014. Generation of mouse induced pluripotent stem cells by protein transduction. Tissue Eng. Part C Methods 20, 383–392. Okita, K., Hong, H., Takahashi, K., Yamanaka, S., 2010. Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5, 418–428. Pan, C., Lu, B., Chen, H., Bishop, C.E., 2010. Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Mol. Biol. Rep. 37, 2117–2124. Rajendra, Y., Kiseljak, D., Baldi, L., Hacker, D.L., Wurm, F.M., 2011. A simple high-yielding process for transient gene expression in CHO cells. J. Biotechnol. 153, 22–26. Rasmussen, S.G.F., Choi, H.J., Rosenbaum, D.M., Kobilka, T.S., Thian, F.S., Edwards, P.C., Burghammer, M., Ratnala, V.R.P., Sanishvili, R., Fischetti, R.F., Schertler, G.F.X., Weis, W.I., Kobilka, B.K., 2007. Crystal structure of the human beta2 adrenergic G-proteincoupled receptor. Nature 450, 383–387. Shen, X., Sanishvili, R., Hacker, D.L., Baldi, L., Wurm, F.M., 2014. Virus-free transient protein production in Sf9 cells. J. Biotechnol. 171, 61–70. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., Hochedlinger, K., 2008. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Vijaya, Gopal, 2013. Bioinspired peptides as versatile nucleic acid delivery platforms. J. Control. Release 167, 323–332. White, J.F., Noinaj, N., Shibata, Y., Love, J., Kloss, B., Xu, F., Gvozdenovic-Jeremic, J., Shah, P., Shiloach, J., Tate, C.G., Grisshammer, R., 2012. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513. Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P.T., Gertsenstein, M., 2009. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770. Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I.I., Thomson, J.A., 2009. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801. Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J., Jin, Y., 2012. Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials 33, 5047–5055. Zhou, H.Y., Wu, S.L., Joo, J.Y., Zhu, S.Y., Han, D.W., Lin, T.X., Trauger, S., Bien, G., Yao, S., Zhu, Y., 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384.
Please cite this article as: Pan, C., et al., Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.061