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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074
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
Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91(2):192–206
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
Dahlroth SL, Nordlund P, Cornvik T (2006) Colony filtration blotting for screening soluble expression in Escherichia coli. Nat Protoc 1(1):253–258
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
Festa F, Steel J, Bian X, Labaer J (2013) High-throughput cloning and expression library creation for functional proteomics. Proteomics 13(9):1381–1399
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
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
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
Ishida T, Kinoshita K (2008) Prediction of disordered regions in proteins based on the meta approach. Bioinformatics 24(11):1344–1348
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
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
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
Razinia Z, Makela T, Ylanne J, Calderwood DA (2012) Filamins in mechanosensing and signaling. Ann Rev Biophys 41:227–246
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
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
Striegel AM, Brewer AK (2012) Hydrodynamic chromatography. Ann Rev Anal Chem 5:15–34
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234
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
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
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
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.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-014-1699-x