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

Amino Acids volume 46, pages 1565–1582 (2014)Cite this article

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.

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References

  • Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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):447–450

    CAS  PubMed  Article  Google Scholar 

  • Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91(2):192–206

    CAS  PubMed  Article  Google Scholar 

  • 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):9062–9065

    CAS  PubMed  Google Scholar 

  • Dahlroth SL, Nordlund P, Cornvik T (2006) Colony filtration blotting for screening soluble expression in Escherichia coli. Nat Protoc 1(1):253–258

    CAS  PubMed  Article  Google Scholar 

  • 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

  • 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

  • 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):915–919

    CAS  PubMed  Article  Google Scholar 

  • Festa F, Steel J, Bian X, Labaer J (2013) High-throughput cloning and expression library creation for functional proteomics. Proteomics 13(9):1381–1399

    CAS  PubMed  Article  Google Scholar 

  • 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):1021–1032

    CAS  PubMed  Article  Google Scholar 

  • 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):10328–10338

    CAS  PubMed  Article  Google Scholar 

  • 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):135–146

  • 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):210–221

    PubMed  Article  Google Scholar 

  • Ishida T, Kinoshita K (2008) Prediction of disordered regions in proteins based on the meta approach. Bioinformatics 24(11):1344–1348

    CAS  PubMed  Article  Google Scholar 

  • 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–W405

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • 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):2212–2221

    CAS  PubMed  Article  Google Scholar 

  • 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):221–227

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Razinia Z, Makela T, Ylanne J, Calderwood DA (2012) Filamins in mechanosensing and signaling. Ann Rev Biophys 41:227–246

    CAS  Article  Google Scholar 

  • 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):209–214

    CAS  PubMed  Article  Google Scholar 

  • 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):138–145

    CAS  PubMed  Article  Google Scholar 

  • Striegel AM, Brewer AK (2012) Hydrodynamic chromatography. Ann Rev Anal Chem 5:15–34

    CAS  Article  Google Scholar 

  • Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234

    CAS  PubMed  Article  Google Scholar 

  • 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):165–172

    CAS  PubMed  Article  Google Scholar 

  • 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):65–72

    CAS  PubMed  Article  Google Scholar 

  • 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):601–605

    CAS  PubMed  Article  Google Scholar 

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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.

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Author notes

  1. Nikos Pinotsis

    Present address: Birkbeck College, University of London, Malet Street, London, WC1E 7HX, UK

  2. Anita Lehner, Jana Neuhold & Peggy Stolt-Bergner

    Present address: Protein Technologies Facility, Campus Science Support Facilities GmbH, Dr. Bohrgasse 3, 1030, Vienna, Austria

Authors and Affiliations

  1. Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria

    Georg Mlynek, Anita Lehner, Jana Neuhold, Sarah Leeb, Julius Kostan, Alexej Charnagalov, Kristina Djinović-Carugo & Nikos Pinotsis

  2. Research Institute of Molecular Pathology, Dr. Bohr-gasse 7, 1030, Vienna, Austria

    Peggy Stolt-Bergner

  3. Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000, Ljubljana, Slovenia

    Kristina Djinović-Carugo

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  1. Georg Mlynek
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  2. Anita Lehner
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Correspondence to Kristina Djinović-Carugo or Nikos Pinotsis.

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Mlynek, G., Lehner, A., Neuhold, J. et al. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein–protein complexes. Amino Acids 46, 1565–1582 (2014). https://doi.org/10.1007/s00726-014-1699-x

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  • DOI: https://doi.org/10.1007/s00726-014-1699-x

Keywords

  • Construct optimization
  • Ligation-indepedent cloning
  • Expression screening
  • Optimized protein production
  • Structural biology