A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts

Autoři: Márk Somogyi aff001;  Tamás Szimler aff001;  Attila Baksa aff001;  Barbara M. Végh aff001;  Tamás Bakos aff001;  Katalin Paréj aff001;  Csaba Ádám aff001;  Áron Zsigmond aff001;  Márton Megyeri aff001;  Beáta Flachner aff001;  Ráchel Sajó aff001;  Éva Gráczer aff001;  Péter Závodszky aff001;  István Hajdú aff001;  László Beinrohr aff001
Působiště autorů: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pázmány Péter sétány, Budapest, Hungary aff001
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0227110


We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

Klíčová slova:

Insect vectors – Proteases – Protein expression – Protein kinases – Recombinant proteins – Secretion – Yeast – Pichia pastoris


1. Aricescu AR, Lu W, Jones EY. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Cryst D. 2006;62:1243–50.

2. Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achivements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014;98:5301–17. doi: 10.1007/s00253-014-5732-5 24743983

3. Zitzmann J, Sprick G, Weidner T, Schreiber C, Czermak P. Process optimization for recombinant protein expression in insect cells. In: Gowder SJT, editor. New insights into cell culture technology. 1. London, UK: InTechOpen; 2017. p. 43–97.

4. Lyska D, Engelmann K, Meierhoff K, Westhoff P. pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis. PLoS One. 2013;8(1):e53787. doi: 10.1371/journal.pone.0053787 23326506

5. Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, Arolas JL, et al. The pCri system: A vector collection for recombinant protein expression and purification. PloS One. 2014;9(11):e112643. doi: 10.1371/journal.pone.0112643 25386923

6. Deng X, Zhang G, Zhang L, Feng Y, Li Z, Wu G, et al. Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA. PloS One. 2015;10(11):e0142558. doi: 10.1371/journal.pone.0142558 26556098

7. Martin CD, Rojas G, Mitchel JN, Vincent KJ, Wu J, McCafferty J, et al. A simple vector system to improve performance and utilisation of recombinant antibodies. BMC Biotechnology. 2006;6:46. doi: 10.1186/1472-6750-6-46 17156422

8. Fang F, Salmon K, Shen MWY, Aeling KA, Ito E, Irwin B, et al. A vector set for systematic metabolic engineering in Saccharomyces cerevisiae. Yeast. 2011;28(2):123–36. doi: 10.1002/yea.1824 20936606

9. Koch JC, Tatenhorst L, Roser A-E, Saal K-A, Tönges L, Lingor P. ROCK inhibition in models of neurodegeneration and its potential for clinical translation. Pharmacol Ther. 2018;189:1–21. doi: 10.1016/j.pharmthera.2018.03.008 29621594

10. Truebestein L, Elsner DJ, Fuchs E, Leonard TA. A moleculer ruler regulates cytoskeletal remodelling by the Rho kinases. Nat Commun. 2015;6:10029. doi: 10.1038/ncomms10029 26620183

11. Hirz M, Richter G, Leitner E, Wriessnegger T, Pichler H. A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform. Appl Microbiol Biotechnol. 2013;97:9465–78. doi: 10.1007/s00253-013-5156-7 23955473

12. Scott RW, Olson MF. LIM kinases: function, regulation and association with human disease. J Mol Med. 2007;85:555–68. doi: 10.1007/s00109-007-0165-6 17294230

13. Yan M, Wang C, He B, Yang M, Tong M, Long Z, et al. Aurora-A kinase: A potent oncogene and target for cancer theraphy. Med Res Rev. 2016;36(6):1036–79. doi: 10.1002/med.21399 27406026

14. Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci. 2007;120(Pt 18):3163–72. Epub 2007/09/20. 120/18/3163 [pii] doi: 10.1242/jcs.010389 17878233.

15. Amin KS, Banerjee PP. The cellular functions of RASSF1A and its inactivation in prostate cancer. J Carcinog. 2012;11(3):31–8. Epub 2012/03/23. doi: 10.4103/1477-3163.93000 JC-11-3 [pii]. 22438769.

16. Chen B-Y, Janes HW. PCR cloning protocols: Humana Press; 2002.

17. Green MR, Sambrook J. Molecular cloning: A laboratory manual. 4th ed. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press; 2012.

18. Reeves A. In vitro mutagenesis: Humana press; 2017.

19. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT. Nucleic Acids Symp. 1999;41(1):95–8.

20. NovaGen. pET system manual. 11th ed: Merck; 2006.

21. Loomis KH, Yaeger KW, Batenjany MM, Mehler MM, Grabski AC, Wong SC, et al. InsectDirectTM System rapid, high-level protein expression and purification from insect cells. J Struct Funct Genomics. 2005;6:189–94. doi: 10.1007/s10969-005-5241-y 16211518

22. Invitrogen. Guide to Baculovirus Expression Vector Systems (BEVS) and insect cell culture techniques: Life Technologies Corporation.

23. Invitrogen. Pichia expression kit manual: Life Technologies Corporation; 2014.

24. Novagen. Insect GeneJuice transfection reagent. 2009.

25. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995;4:2411–23. doi: 10.1002/pro.5560041120 8563639

26. Schneider CA, Rasband WS, Eliceiri KW. NIH to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5. doi: 10.1038/nmeth.2089 22930834

27. Rodems SM, Hamman BD, Lin C, Zhao J, Shah S, Heidary D, et al. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol. 2002;1(1–1):9–19.

28. Invitrogen. Z'-LyteTM kinase assay kit. 2005.

29. Bach H, Mazor Y, Shaky S, Shoham-Lev A, Berdichevsky Y, Guttnick DL, et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol. 2001;312(1):79–93. doi: 10.1006/jmbi.2001.4914 11545587

30. Kapust RB, Tözsér J, Copeland TD, Waugh DS. The P1' specificity of tobacco etch virus protease. Biochem Biophys Res Commun. 2002;294(5):949–55. doi: 10.1016/S0006-291X(02)00574-0 12074568

31. Cabrita LD, Gilis D, Robertson AL, Dehouck Y, Rooman M, Bottomley SP. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 2007;16(11):2360–7. doi: 10.1110/ps.072822507 17905838

32. Pustelny K, Zdzalik M, Stach N, Stec-Niemczyk J, Cichon P, Czarna A, et al. Staphylococcal SpIB serine protease utilizes a novel molecular mechanism of activation. J Biol Chem. 2014;289(22):15544–53. doi: 10.1074/jbc.M113.507616 24713703

33. Dälken B, Jabulowsky RA, Oberoi P, Benhar I, Wels WS. Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein. PloS One. 2010;5(12):e14404. doi: 10.1371/journal.pone.0014404 21203542

34. Scholz J, Besir H, Strasser C, Suppmann S. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning. BMC Biotechnology. 2013;13:12. doi: 10.1186/1472-6750-13-12 23410102

35. Bokhove M, Sadat Al Hosseini H, Saito T, Dioguardi E, Gegenschatz-Schmid K, Nishimura K, et al. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins. J Struct Biol. 2016;194(1):1–7. doi: 10.1016/j.jsb.2016.01.016 26850170

36. Reuten R, Nikodemus D, Oliveira MB, Patel TR, Brachvogel B, Breloy I, et al. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems. PloS One. 2016;11(3):e0152386. doi: 10.1371/journal.pone.0152386 27029048

37. Herskowitz JH, Feng Y, Mattheyses AL, Hales CM, Higginbotham LA, Duong DM, et al. Pharmacologic Inhibition of ROCK2 Suppresses Amyloid-B Production in an Alzheimer’s Disease Mouse Model. J Neurosci. 2013;33:19086–98. doi: 10.1523/JNEUROSCI.2508-13.2013 24305806

38. Szimler T, Gráczer É, Györffy D, Végh B, Szilágyi A, Hajdú I, et al. New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and it mitotic kinase Aurora A. Sci Rep. 2019;9:5550. doi: 10.1038/s41598-019-41972-x 30944388

39. Balogh A, Toth E, Romero R, Parej K, Csala D, Szenasi NL, et al. Placental galectins are key players in regulating the maternal adaptive immune response. Front Immunol. 2019. doi: 10.3389/fimmu.2019.01240 31275299

40. Mirmazloum I, Ladányi M, Beinrohr L, Kiss-Bába E, Kiss A, György Z. Identification of a novel UDP-glycosyltransferase gene from Rhodiola rosea and its expression during biotransformation of upstream precursors in callus culture. Int J Macromol. 2019;136:847–58.

41. Laitinen OH, Airenne KJ, Hytönen VP, Peltomaa E, Mähönen AJ, Wirth T, et al. A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo. Nucleic Acids Res. 2005;33(4):e42. doi: 10.1093/nar/gni042 15731335

42. Sinah N, Williams CA, Piper RC, Shields SB. A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid. BMC Biotechnology. 2012;12:54. doi: 10.1186/1472-6750-12-54 22916790

Článok vyšiel v časopise


2019 Číslo 12