Amino Acids

, Volume 46, Issue 6, pp 1565–1582 | Cite as

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein–protein complexes

  • Georg Mlynek
  • Anita Lehner
  • Jana Neuhold
  • Sarah Leeb
  • Julius Kostan
  • Alexej Charnagalov
  • Peggy Stolt-Bergner
  • Kristina Djinović-Carugo
  • Nikos Pinotsis
Amino Acids Protocols
First Online:
Received:
Accepted:

Abstract

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20–100 different constructs followed by large-scale purification of at least 4–6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Keywords

Construct optimization Ligation-indepedent cloning Expression screening Optimized protein production Structural biology 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This project was supported by the Federal Ministry of Economy, Family and Youth through the initiative “Laura Bassi Centres of Expertise”, funding Center of Optimized Structural Studies, project Number 253275; JK was also supported by the Austrian Science Fund (FWF) Project P22276. FWF Doctoral program BioToP-Molecular Technology of Proteins (W1224) is acknowledged to providing training for GM. We thank Dr. Nathalie Landstetter at QIAGEN for generously providing the use of the instrument and valuable suggestions and help with experiments.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1699_MOESM1_ESM.rtf (3.6 mb)
Supplementary material 1 (RTF 3678 kb)
726_2014_1699_MOESM2_ESM.wmp (24 kb)
Supplementary material 2 (WMP 23 kb)

References

  1. Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074PubMedCentralPubMedCrossRefGoogle Scholar
  2. Bieniossek C, Nie Y, Frey D, Olieric N, Schaffitzel C, Collinson I, Romier C, Berger P, Richmond TJ, Steinmetz MO, Berger I (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6(6):447–450PubMedCrossRefGoogle Scholar
  3. Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91(2):192–206PubMedCrossRefGoogle Scholar
  4. Cordingley MG, Callahan PL, Sardana VV, Garsky VM, Colonno RJ (1990) Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J Biol Chem 265(16):9062–9065PubMedGoogle Scholar
  5. Dahlroth SL, Nordlund P, Cornvik T (2006) Colony filtration blotting for screening soluble expression in Escherichia coli. Nat Protoc 1(1):253–258PubMedCrossRefGoogle Scholar
  6. Djinović-Carugo K, Pinotsis N (2013) Automated protein expression and subsequent protein purification screen. http://www.mynewsdesk.com/de/pressroom/hamilton-robotics/pressrelease/view/automatisiertes-screening-von-small-scale-proteinen-906051
  7. Djinović-Carugo K, Pinotsis N, Charnagalov A, Kostan J, Mlynek G (2013) Automation of the Macherey-Nagel NucleoFast PCR Cleanup Kit and the QIAprep® 96Plus Biorobot® on a Hamilton Microlab STARlet Liquid Handler. http://www.mynewsdesk.com/de/pressroom/hamilton-robotics/pressrelease/view/automation-des-macherey-nagel-nucleofast-pcr-cleanup-kits-und-des-qiaprep-r-96plus-biorobot-r-911245
  8. Dupeux F, Rower M, Seroul G, Blot D, Marquez JA (2011) A thermal stability assay can help to estimate the crystallization likelihood of biological samples. Acta Crystallogr D Biol Crystallogr 67(Pt 11):915–919PubMedCrossRefGoogle Scholar
  9. Festa F, Steel J, Bian X, Labaer J (2013) High-throughput cloning and expression library creation for functional proteomics. Proteomics 13(9):1381–1399PubMedCrossRefGoogle Scholar
  10. Fitzgerald DJ, Berger P, Schaffitzel C, Yamada K, Richmond TJ, Berger I (2006) Protein complex expression by using multigene baculoviral vectors. Nat Methods 3(12):1021–1032PubMedCrossRefGoogle Scholar
  11. Gerard FC, Ribeiro Ede A Jr, Albertini AA, Gutsche I, Zaccai G, Ruigrok RW, Jamin M (2007) Unphosphorylated rhabdoviridae phosphoproteins form elongated dimers in solution. Biochemistry 46(36):10328–10338PubMedCrossRefGoogle Scholar
  12. Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008a) Protein production and purification. Nat Methods 5(2):135–146Google Scholar
  13. Graslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarstrom M, Johansson I, Kotenyova T, Nilsson M, Nordlund P, Weigelt J (2008b) The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr Purif 58(2):210–221PubMedCrossRefGoogle Scholar
  14. Ishida T, Kinoshita K (2008) Prediction of disordered regions in proteins based on the meta approach. Bioinformatics 24(11):1344–1348PubMedCrossRefGoogle Scholar
  15. Mooij WT, Mitsiki E, Perrakis A (2009) ProteinCCD: enabling the design of protein truncation constructs for expression and crystallization experiments. Nucleic Acids Res 37((Web Server issue)):W402–W405PubMedCentralPubMedCrossRefGoogle Scholar
  16. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2(9):2212–2221PubMedCrossRefGoogle Scholar
  17. Radivojac P, Clark WT, Oron TR, Schnoes AM, Wittkop T, Sokolov A, Graim K, Funk C, Verspoor K, Ben-Hur A, Pandey G, Yunes JM, Talwalkar AS, Repo S, Souza ML, Piovesan D, Casadio R, Wang Z, Cheng J, Fang H, Gough J, Koskinen P, Toronen P, Nokso-Koivisto J, Holm L, Cozzetto D, Buchan DW, Bryson K, Jones DT, Limaye B, Inamdar H, Datta A, Manjari SK, Joshi R, Chitale M, Kihara D, Lisewski AM, Erdin S, Venner E, Lichtarge O, Rentzsch R, Yang H, Romero AE, Bhat P, Paccanaro A, Hamp T, Kassner R, Seemayer S, Vicedo E, Schaefer C, Achten D, Auer F, Boehm A, Braun T, Hecht M, Heron M, Honigschmid P, Hopf TA, Kaufmann S, Kiening M, Krompass D, Landerer C, Mahlich Y, Roos M, Bjorne J, Salakoski T, Wong A, Shatkay H, Gatzmann F, Sommer I, Wass MN, Sternberg MJ, Skunca N, Supek F, Bosnjak M, Panov P, Dzeroski S, Smuc T, Kourmpetis YA, van Dijk AD, ter Braak CJ, Zhou Y, Gong Q, Dong X, Tian W, Falda M, Fontana P, Lavezzo E, Di Camillo B, Toppo S, Lan L, Djuric N, Guo Y, Vucetic S, Bairoch A, Linial M, Babbitt PC, Brenner SE, Orengo C, Rost B, Mooney SD, Friedberg I (2013) A large-scale evaluation of computational protein function prediction. Nat Methods 10(3):221–227PubMedCentralPubMedCrossRefGoogle Scholar
  18. Razinia Z, Makela T, Ylanne J, Calderwood DA (2012) Filamins in mechanosensing and signaling. Ann Rev Biophys 41:227–246CrossRefGoogle Scholar
  19. Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J, Weiss MS (2013) Optimization of protein buffer cocktails using Thermofluor. Acta Crystallogr Sect F Struct Biol Cryst Commun 69(Pt 2):209–214PubMedCrossRefGoogle Scholar
  20. Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, Shapiro SS (2001) Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2(2):138–145PubMedCrossRefGoogle Scholar
  21. Striegel AM, Brewer AK (2012) Hydrodynamic chromatography. Ann Rev Anal Chem 5:15–34CrossRefGoogle Scholar
  22. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234PubMedCrossRefGoogle Scholar
  23. Vincentelli R, Bignon C, Gruez A, Canaan S, Sulzenbacher G, Tegoni M, Campanacci V, Cambillau C (2003) Medium-scale structural genomics: strategies for protein expression and crystallization. Acc Chem Res 36(3):165–172PubMedCrossRefGoogle Scholar
  24. Vincentelli R, Cimino A, Geerlof A, Kubo A, Satou Y, Cambillau C (2011) High-throughput protein expression screening and purification in Escherichia coli. Methods 55(1):65–72PubMedCrossRefGoogle Scholar
  25. Walker PA, Leong LE, Ng PW, Tan SH, Waller S, Murphy D, Porter AG (1994) Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology 12(6):601–605PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Georg Mlynek
    • 1
  • Anita Lehner
    • 1
    • 5
  • Jana Neuhold
    • 1
    • 5
  • Sarah Leeb
    • 1
  • Julius Kostan
    • 1
  • Alexej Charnagalov
    • 1
  • Peggy Stolt-Bergner
    • 2
    • 5
  • Kristina Djinović-Carugo
    • 1
    • 3
  • Nikos Pinotsis
    • 1
    • 4
  1. 1.Department of Structural and Computational BiologyMax F. Perutz Laboratories, University of ViennaViennaAustria
  2. 2.Research Institute of Molecular PathologyViennaAustria
  3. 3.Department of Biochemistry, Faculty of Chemistry and Chemical TechnologyUniversity of LjubljanaLjubljanaSlovenia
  4. 4.Birkbeck CollegeUniversity of LondonLondonUK
  5. 5.Protein Technologies Facility, Campus Science Support Facilities GmbHViennaAustria

Personalised recommendations