改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白
Summary
在这里, 我们提出了利用昆虫细胞和杆状病毒蛋白表达系统来产生大量植物分泌蛋白的蛋白质结晶的协议。用 GP67 或昆虫 hemolin 信号肽对昆虫细胞中植物蛋白分泌表达进行了改进, 并对杆状病毒表达载体进行了修正。
Abstract
对科学家来说, 表达重组分泌真核蛋白用于结构和生物化学研究是一个挑战。杆状病毒介导的昆虫细胞表达系统是用于表达重组真核分泌蛋白的系统之一, 具有一定的转化后修饰。分泌蛋白需要通过分泌通路进行蛋白质糖基化、二硫化键形成和其他转化后的修饰。为改善现有分泌植物蛋白的昆虫细胞表达, 在启动子和多克隆点之间添加 GP67 或 hemolin 信号肽序列, 对杆状病毒表达载体进行修改。新设计的改进后的载体系统成功地生产出了拟南芥可溶性重组分泌植物受体蛋白的高产。对两种表达的植物蛋白, 拟南芥和 PRK3 膜受体的胞外域进行了结晶, 用于 x 射线晶体的研究。改进后的矢量系统是一种改良的工具, 可用于动物王国中重组分泌蛋白的表达。
Introduction
研究实验室有能力生产大量的均质重组蛋白, 用于生化和生物物理特征, 尤其是 x 射线晶体学研究。大肠杆菌、酵母、昆虫细胞、哺乳动物细胞、植物细胞等具有良好的外源表达系统, 其中杆状病毒介导的昆虫细胞表达系统是其中最常用的技术生产大量的结构折叠大型重组真核蛋白的蛋白质结晶1。
对杆状病毒表达系统的表达载体进行了设计, 以包含强多角体或 P10 启动子, 以产生高产量的重组细胞内蛋白2,3。为了制作重组杆状病毒, 该基因被克隆成含有杆状使用杆状多 nucleopolyhedroviral 基因组多角体 (polh) 轨迹的昆虫载体。然后对生成的构造进行排序, 验证其正确的开放阅读框架 (ORF)。通过转染过程将正确的构造引入宿主昆虫细胞。兴趣基因通过同源重组插入病毒基因组。这一事件导致重组病毒基因组的生产, 然后复制, 以产生重组发芽病毒粒子1。
在表达系统中最常用的昆虫细胞是 Sf9 和高五 (Hi5) 细胞。Sf9 细胞是 Sf21 蛹卵巢细胞的克隆分离物, Hi5 细胞是由父母夜蛾镍卵巢细胞系 TN-3684,5所衍生的克隆分离株。transfections、病毒扩增和斑块检测是在 Sf9 细胞上进行的, 而 Hi5 细胞通常被选择以产生更高数量的重组蛋白6。值得注意的是, Hi5 细胞由于其产生突变病毒的倾向而不适合于病毒后代的产生和扩增。传统上, 25-30 °c 的温度范围被认为对昆虫细胞的培养是有益的。然而, 据报道, 27-28 °c 是最佳的温度为昆虫细胞生长和传染7,8。
在基因之前引入一个强烈的信号序列是分泌蛋白的高表达所必需的。该信号序列将有效地引导转化后的重组蛋白转化为内质网的蛋白质分泌和转化后的修改必要的适当折叠和稳定3。信号肽序列, 如杆状病毒包络蛋白 GP64/67, 蜜蜂蜂毒等, 已被选择, 以促进分泌重组蛋白的表达在杆状病毒介导的表达系统3。GP67 的信号肽的引入, 以提高分泌重组蛋白的表达率, 与使用目标基因9的内在信号肽相比较。Hemolin 是大丝蛾Hyalophora cecropia的血淋巴蛋白, 引起细菌感染10。由于诱导表达水平较高, 该基因的信号肽序列可以用来调节杆状病毒-昆虫细胞系统中重组蛋白的分泌表达。
Tracheary 元素分化抑制因子受体 (TDR) 和花粉受体激酶 3 (PRK3) 都属于植物亮氨酸丰富的重复受体样激酶 (LRR RLK) 蛋白11,12 的家庭.为了研究植物受体蛋白家族的结构和功能, 以及促进其他植物分泌蛋白的结构和生化特性, 对杆状病毒-昆虫细胞表达系统进行了改进, 以提高蛋白质质量和产量。利用两种修饰表达载体在杆状病毒-昆虫细胞表达系统中成功地表达了 TDR 和 PRK3 的胞外域。TDR 和 PRK3 蛋白的胞外域均已结晶。本文报道了两种改进的杆状病毒表达载体的大量重组分泌植物蛋白的表达和纯化, 通过在启动子和多克隆之间加入 GP67 或 hemolin 信号序列。网站。
Protocol
注: 采用修饰表达载体的昆虫细胞/杆状病毒系统, 用于分泌植物蛋白的表达和结晶。 1. 用 GP67 信号肽对杆状病毒表达载体进行植物蛋白分泌表达的改进 合成一个 DNA 片段, 包含 5 ‘ BglII 切割站点13, GP67 分泌信号序列, 和一个多克隆网站与 NotI, BamHI, EcoRI, StuI, 贝里沙, SpeI, XbaI, PstI 和 XhoI (图 1A)。注: 由于 BglII 和 BamHI 消化 dna 导致?…
Representative Results
如图 1所示, 两种改进的 pFastBac1 杆状病毒表达载体用于表达 GP67 或 hemolin 信号序列的分泌蛋白, 以取代目标基因的内在信号序列。病毒 GP67 和昆虫 hemolin 基因已被证明具有较高的分泌表达水平的细胞。融合蛋白与这两个信号序列中的任何一个有望大大提高分泌表达水平。在这两种改进的向量中, 都设计了相同的多个克隆站点 (MCS)。NotI 站点被蓄意放置?…
Discussion
鉴于生物系统中数以千计的蛋白质的大小和稳定性的多样性, 研究实验室通常有经验来决定哪些异种表达系统必须选择用于表达特定的蛋白质。大肠杆菌表达系统往往是由于细菌生命周期短、培养基成本低、相对容易扩展19而导致蛋白质表达的首选。然而, 对于大小超过 60 kDa 的大真核蛋白的表达, 使用大肠杆菌系统往往导致不溶性蛋白在包涵体或聚集20<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自北卡罗来纳州立大学 Guozhou 许的新的教员启动基金的支持。
Materials
| Incubator shaker | VWR | Model Excella E25 | 27 oC |
| Incubator | VWR | Model 2005 | 27 oC |
| Centrifuge | BECKMAN | Model J-6 | with a swing bucket rotor |
| Herculase II Fusion DNA Polymerase | Agilent | 600677-51 | For PCR amplification of DNA |
| Thermal Cycler | BIO-RAD | Model C1000 Touch | For PCR amplification of DNA |
| Incubator shaker | New Brunswick Scientific | Model I 24 | For growing baceria culture |
| Customer DNA synthesis | GENSCRIPT | ||
| BamHI (HF) | New England Biolabs | R3136S | Restriction Endonuclease |
| BglII | New England Biolabs | R0144S | Restriction Endonuclease |
| NotI (HF) | New England Biolabs | R3189S | Restriction Endonuclease |
| XhoI | New England Biolabs | R0146S | Restriction Endonuclease |
| T4 DNA ligase | New England Biolabs | M0202T | DNA ligation |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | DNA purification from Agorase gel |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | Plasmid DNA purification from bacteria culture |
| Agarose | Thermo Fisher Scientific | 15510-019 | For DNA gel electropherosis |
| MAX Efficiency DH5α competen cell | Invitrogen | 18-258-012 | For transformation of DNA ligation mixture |
| Lennox L LB Broth | Research Product International Corp. | L24066-5000.0 | For making bacteria culture |
| Ampicillin sodium salt | Thermo Fisher Scientific | 11593-019 | Antibiotics |
| Kanamycin Sulfate | Thermo Fisher Scientific | 15160-054 | Antibiotics |
| Tetracycline | Thermo Fisher Scientific | 64-75-5 | Antibiotics |
| Gentamicin | Thermo Fisher Scientific | 15710-064 | Antibiotics |
| MAX Efficiency DH10Bac competent cells | Thermo Fisher Scientific | 10361-012 | For making bacmid DNA |
| S.O.C. Medium | Thermo Fisher Scientific | 15544-034 | For DNA transformation |
| CellFECTIN II Reagent | Thermo Fisher Scientific | 10362-101 | Insect cell transfection reagent |
| Bac-to-Bac Expression System | Thermo Fisher Scientific | 10359-016 | Baculovirus-insect cells expression kit |
| Bluo-gal | Thermo Fisher Scientific | 15519-028 | For isolation of recominant Bacmid DNA |
| IPTG | Thermo Fisher Scientific | 15529-019 | For isolation of recominant Bacmid DNA |
| pFastBac1 | Thermo Fisher Scientific | 10360014 | Baculorirus expression vector |
| Sf9 cells | Thermo Fisher Scientific | 11496015 | Sf9 monolayer cells |
| Hi5 cells | Thermo Fisher Scientific | B85502 | High Five insect cells |
| Grace’s insect medium, unsupplemented | Thermo Fisher Scientific | 11595030 | Sf9 cell transfection minimum medium |
| Grace’s insect medium, supplemented | Thermo Fisher Scientific | 11605102 | Sf9 monolayer cell culture complete medium |
| Sf-900 II SFM | Thermo Fisher Scientific | 10902104 | Sf9 suspension cell culture medium without FBS |
| Express Five SFM | Thermo Fisher Scientific | 10486025 | Hi5 cell culture medium |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | 100 ml (10,000 I.U./ml) |
| L-Glutamine (200 mM) | Thermo Fisher Scientific | 25030081 | 100 ml |
| FBS Certified | Thermo Fisher Scientific | 16000-044 | 500 ml |
| 6-well plates | Thermo Fisher Scientific | 08-772-1B | Flat-bottom |
| 150 mm plates | Thermo Fisher Scientific | 353025 | 100/case |
| 1.5 ml Microcentrifuge Tubes | USA Scientific | 1415-2500 | 500 tubes/bag |
| 15 ml conical screw cap centrifuge tubes | USA Scientific | 1475-0511 | 25 tubes/bag |
| 50 ml conical screw cap centrifuge tubes | USA Scientific | 1500-1211 | 25 tubes/bag |
| Ni-NTA Superflow | Qiagen | 30430 | NiNTA resin |
| pH-indicator strips | EMD Millipore Corporation | 1.09535.0001 | pH 0 – 14 |
References
- Contreras-Gomez, A., Sanchez-Miron, A., Garcia-Camacho, F., Molina-Grima, E., Chisti, Y. Protein production using the baculovirus-insect cell expression system. Biotechnology Progress. 30, 1-18 (2014).
- Khan, S., Ng, M. L., Tan, Y. J. Expression of the severe acute respiratory syndrome coronavirus 3a protein and the assembly of coronavirus-like particles in the baculovirus expression system. Methods in Molecular Biology. 379, 35-50 (2007).
- Possee, R. D., King, L. A. Baculovirus Transfer Vectors. Methods in Molecular Biology. 1350, 51-71 (2016).
- Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., McCawley, P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro. 13, 213-217 (1977).
- Hink, W. F. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature. 226, 466-467 (1970).
- Davis, T. R., et al. Comparative recombinant protein production of eight insect cell lines. In Vitro Cellular & Development Biology – Animal. 29A, 388-390 (1993).
- Wickham, T. J., Nemerow, G. R. Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system. Biotechnology Progress. 9, 25-30 (1993).
- Davis, T. R., Trotter, K. M., Granados, R. R., Wood, H. A. Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion. Nature Biotechnology. 10, 1148-1150 (1992).
- Murphy, C. I., et al. Enhanced expression, secretion, and large-scale purification of recombinant HIV-1 gp120 in insect cell using the baculovirus egt and p67 signal peptides. Protein Expression and Purification. 4, 349-357 (1993).
- Sun, S. C., Lindstrom, I., Boman, H. G., Faye, I., Schmidt, O. Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily. Science. 250, 1729-1732 (1990).
- Li, Z., Chakraborty, S., Xu, G. Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions. PLoS One. 12, e0175317 (2017).
- Chakraborty, S., Pan, H., Tang, Q., Woolard, C., Xu, G. The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors. Scientific Reports. 8, 2796 (2018).
- Khorana, H. G. Total synthesis of a gene. Science. 203, 614-625 (1979).
- Bahl, C. P., Marians, K. J., Wu, R. A general method for inserting specific DNA sequences into cloning vehicles. Gene. 1, 81-92 (1976).
- Xu, W., Zhang, Y. Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative. Methods in Molecular Biology. 1387, 213-226 (2016).
- Hanahan, D. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology. 166, 557-580 (1983).
- Zhang, S., Cahalan, M. D. Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit. Journal of Visualized Experiments. (6), 247 (2007).
- Reddy, B. G., et al. Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC. Protein Expression and Purification. 117, 67-75 (2016).
- Harris, T. J., Emtage, J. S. Expression of heterologous genes in E. coli. Microbiological Sciences. 3, 28-31 (1986).
- Kaur, J., Kumar, A., Kaur, J. Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. International Journal of BIological Macromolecules. 106, 803-822 (2018).
- Guo, Y., Yang, F., Tang, X. An Overview of Protein Secretion in Yeast and Animal Cells. Methods in Molecular Biology. 1662, 1-17 (2017).
- Blight, M. A., Chervaux, C., Holland, I. B. Protein secretion pathway in Escherichia coli. Current Opinion in Biotechnology. 5, 468-474 (1994).
- Idiris, A., Tohda, H., Kumagai, H., Takegawa, K. Engineering of protein secretion in yeast: strategies and impact on protein production. Applied Microbiology and Biotechnology. 86, 403-417 (2010).
- Wormald, M. R., Dwek, R. A. Glycoproteins: glycan presentation and protein-fold stability. Structure. 7, R155-R160 (1999).
- Geisler, C., Mabashi-Asazuma, H., Jarvis, D. L. An Overview and History of Glyco-Engineering in Insect Expression Systems. Methods in Molecular Biology. 1321, 131-152 (2015).
- Li, Z., Chakraborty, S., Xu, G. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa. Acta Crystallographica Section F: Structural BIology and Crystallization Communications. 72, 782-787 (2016).
- Aricescu, A. R., Owens, R. J. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Current Opinion in Structural Biology. 23, 345-356 (2013).
- Reuter, L. J., Bailey, M. J., Joensuu, J. J., Ritala, A. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. Plant Biotechnology Journal. 12, 402-410 (2014).
- Dohi, K., et al. Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells. Archives of Virology. 151, 1075-1084 (2006).
Cite This Article
Chakraborty, S., Trihemasava, K., Xu, G. Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells. J. Vis. Exp. (138), e58283, doi:10.3791/58283 (2018).