Protein Technologies

Expertise in recombinant protein technologies and genome engineering

 

The mission of the Protein Technologies Facility (ProTech) is to further research in molecular and cell biology, protein biochemistry, and structural biology by overcoming major bottlenecks in these fields.

Our core services include molecular cloning, protein production and purification, and biophysical characterization of proteins. Since 2014, we also offer services surrounding the CRISPR/Cas9 genome engineering technology.

SERVICES

Molecular Cloning

We offer customized generation of DNA constructs for recombinant protein production as a stand-alone service or as the first step in a protein production project. We are specialized in generating multi-gene expression constructs for production of protein complexes.

Recombinant Protein Production and Purification

We offer recombinant protein production in E. coli, Drosophila S2 cells, insect cells using the baculovirus expression vector system (BEVS), and HEK293 cells.

ProTech uses several optimized baculovirus bacmids, licensed from Geneva Biotech. We can also provide fermentation services through our partner Eucodis Bioscience.

Protein purification is offered as a full service at different scales. Due to limited capacity, for projects requiring purification of multiple proteins we also offer training and access to the equipment in our facility.

Biophysical Characterization

We offer protein characterization services using a variety of Biophysical techniques to study protein stability, oligomeric state, and secondary structure, or to analyze biomolecular interactions.

We also provide instrument training and access for some of these methods. To gain an overview of the techniques available, please see the Equipment section. More information can be found on the ProTech Wiki, accessible via MyVBCF. To discuss your biophysical characterization project, please contact Peggy Stolt-Bergner or Arthur Sedivy.

Genome Engineering

Our facility offers expertise in the CRISPR/Cas9 genome engineering method. Services include consultation and advice on experimental design; preparation of DNA, RNA, and protein reagents; and generation of knock-out and knock-in cell lines or plants.

To discuss your genome engineering project, please contat Vera Schoft (for projects in plants) or Krzysztof Chylinski (for projects in mammalian and other species).

EQUIPMENT

OmniSEC

The OmniSEC system combines analytical size exclusion chromatography with right angle light scattering and measurement of refractive index, to accurately determine protein molecular weights and concentrations. It can be used to analyze the oligomeric state of proteins and protein complexes and to quantify the different species present in a sample. A full UV-vis spectrum and intrinsic viscosity measurements enable the accurate determination of extinction coefficients and shape/size of protein samples.

PEAQ-ITC

The PEAQ-ITC instrument can be used to perform isothermal titration calorimetry experiments to measure biomolecular interaction parameters. The change in enthalpy upon ligand binding is directly measured by the instrument and can then be used to determine the dissociation constant, stoichiometry, and other thermodynamic parameters of the binding interaction. Interactions in the high nanomolar to high micromolar range can be determined using the PEAQ-ITC.

MASS-1

The MASS-1 instrument is a system for measuring biomolecular interactions by Surface Plasmon Resonance (SPR). In addition to determining dissociation constants, SPR is ideal for measuring kinetics of interactions (kon and koff).

The SPR service is only possible as a full-service. In exceptional cases, access is offered to experienced users.

Aekta Purifier

ProTech has three Aekta Purifier/Aekta PURE systems that can be booked by users after appropriate training, so that purifications can be performed by users in our lab. Additional purification equipment, such as a sonicator and centrifuges, can also be booked after training.

Dynapro II DLS Plate Reader

DLS is a label free technique that analyzes particle size distribution in a bulk sample. It is mainly used for protein quality control, as you can detect size distributions of molecules and therefore aggregation and oligomerization of your protein of interest. The Dynapro II plate reader can also be used for screening or long-term stability measurements. Experiments can be carried out for you; alternatively, you may receive training and can then book the instrument yourself.

Chirascan Plus CD Spectrometer

Circular Dichroism is a spectroscopic technique that uses circularly polarized light to study the structure of chiral molecules, such as proteins. The CD spectrum of a protein can be used to determine protein secondary structure or some aspects of tertiary structure. CD can also be used to study protein stability using thermal melt analysis to determine protein melting temperatures (Tm). We perform CD on a Chirascan Plus CD spectrometer from Applied Photophysics.  Experiments can be carried out for you; alternatively, you may receive training and can then book the instrument yourself.

Monolith NT.115 BLUE/RED

MST is a novel technique used to determine molecular affinities. Thermophoretic changes upon molecular interaction can be monitored by fluorescent labeling or via tryptophan fluorescence, revealing binding constants in the range of nM to mM.  Unlike many other methods, MST experiments can be performed rapidly and with small amounts of sample. We have two instruments for performing MST experiments, the NT.115 and the NT.LabelFree. MST experiments can be performed as a service, or you can receive training and then book the instruments yourself.

USER INFORMATION

If you are a new user, please contact Peggy Stolt-Bergner (for ProTech core services) or Krzysztof Chylinski (for genome engineering services) to discuss your project requirements. To order services and book instruments, please use our web-based booking and request system. IMP, IMBA, GMI, and MFPL users can log in with their usual institute network accounts. External users must register. For questions, please contact protech(at)vbcf.ac.at.

Pricing information can be found on the ProTech MyVBCF Wiki site.

The ProTech Facility Usage Policy can be downloaded HERE. It is assumed that users who order services have read and understood this policy as well as the VBCF General Cooperation Conditions. For questions please contact Peggy Stolt-Bergner.

Continuation of funding for VBCF-ProTech from the City of Vienna and the Austrian Federal Ministry of Education, Science, and Research (BMBFW) is dependent on documented evidence of contributions to scientific output. Therefore, please acknowledge use of the facility when publishing work for which ProTech services were used. Please also acknowledge training and instrument access provided by ProTech. A simple statement is sufficient and can either be placed in the Materials and Methods section or in the Acknowledgements.

Suggested format: "ProTech service" was performed by the VBCF Protein Technologies Facility (www.vbcf.ac.at). Your publication will then be listed on our website.

Additional links, information on techniques, prices, and other downloads can be found on the ProTech MyVBCF Wiki site. IMP, IMBA, GMI, and MFPL users can log in with their usual institute network accounts. External users must first register.

PUBLICATIONS

Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions
Julia Richter, James Matthew Watson, Peter Stasnik, Monika Borowska, Jana Neuhold, Matthias Berger, Peggy Stolt-Bergner, Vera Schoft & Marie-Theres Hauser. Scientific Reports volume 8, Article number: 12182 (2018)

Baculovirus-driven protein expression in insect cells: A benchmarking study
Stolt-Bergner P, Benda C, Bergbrede T, Besir H, Celie PHN, Chang C, Drechsel D, Fischer A, Geerlof A, Giabbai B, van den Heuvel J, Huber G, Knecht W, Lehner A, Lemaitre R, Nordén K, Pardee G, Racke I, Remans K, Sander A, Scholz J, Stadnik M, Storici P, Weinbruch D, Zaror I, Lua LHL, Suppmann S. J. Struct. Biol. 2018 Mar 12;

UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins
Hellerschmied D, Roessler M, Lehner A, Gazda L, Stejskal K, Imre R, Mechtler K, Dammermann A, Clausen T. Nat Commun 2018 02 02; 9 (1)

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases
Smakowska-Luzan E, Mott GA, Parys K, Stegmann M, Howton TC, Layeghifard M, Neuhold J, Lehner A, Kong J, Grünwald K, Weinberger N, Satbhai SB, Mayer D, Busch W, Madalinski M, Stolt-Bergner P, Provart NJ, Mukhtar MS, Zipfel C, Desveaux D, Guttman DS, Belkhadir Y. Nature 2018 Jan 18; 553 (7688)

 

Protection of Arabidopsis blunt-ended telomeres is mediated by a physical association with the Ku heterodimer
Valuchova S, Fulnecek J, Prokop Z, Stolt-Bergner P, Janouskova E, Hofr C, Riha K. Plant Cell epub 5 June 2017.

The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling
Stegmann M, Monaghan J, Smakowska-Luzan E, Rovenich H, Lehner A, Holton N, Belkhadir Y, Zipfel C. (2017) Science Jan 20;355(6322):287-289.

Genetic code expansion for multiprotein complex engineering
Koehler C, Sauter PF, Wawryszyn M, Girona GE, Gupta K, Landry JJ, Fritz MH, Radic K, Hoffmann JE, Chen ZA, Zou J, Tan PS, Galik B, Junttila S, Stolt-Bergner P, Pruneri G, Gyenesei A, Schultz C, Biskup MB, Besir H, Benes V, Rappsilber J, Jechlinger M, Korbel JO, Berger I, Braese S, Lemke EA. (2016). Nature Methods 13(12):997-1000 epub Oct 17, 2016

ACKNOWLEDGEMENTS

A Functional Study of AUXILIN-LIKE1 and 2, Two Putative Clathrin Uncoating Factors in Arabidopsis
Maciek Adamowski, Madhumitha Narasimhan, Urszula Kania, Matouš Glanc, Geert De Jaege and Jiří Friml. Plant Cell. 2018 Mar; 30(3): 700–716

Molecular basis for inner kinetochore configuration through RWD domain-peptide interactions
Schmitzberger F, Richter MM, Gordiyenko Y, Robinson CV, Dadlez M, Westermann S. EMBO J. 2017 12 01; 36 (23).

DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes
Samwer M, Schneider MWG, Hoefler R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW. Cell 2017 Aug 24; 170 (5).

HuR small molecule inhibitor elicits differential effects in adenomatosis polyposis and colorectal carcinogenesis
Lang M, Berry D, Passecker K, Mesteri I, Bhuju S, Ebner F, Sedlyarov V, Evstatiev R, Dammann K, Loy A, Kuzyk O, Kovarik P, Khare V, Beibel M, Roma G, Meisner-Kober N and Gasche C. Cancer Research epub 20 Feb 2017.

Root diffusion barrier control by a vasculature-derived peptide binding to the SGN3 receptor
Doblas VG, Smakowska-Luzan E, Fujita S, Alassimone J, Barberon M, Madalinski M, Belkhadir Y, Geldner N. (2017) Science Jan 20;355(6322):280-284.

Probing an Allosteric Pocket of CDK2 with Small Molecules
Christodoulou MS, Caporuscio F, Restelli V, Carlino L, Cannazza G, Costanzi E, Citti C, Lo Presti L, Pisani P, Battistutta R, Broggini M, Passarella D, Rastelli G. ChemMedChem 2017 Jan 05; 12 (1).

A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement
Valuchova S, Fulnecek J, Petrov AP, Tripsianes K, Riha K. (2016). Sci Rep. 6:39653.

Cross-regulation by CrcZ RNA controls anoxic biofilm formation in Pseudomonas aeruginosa
Pusic P, Tata M, Wolfinger MT, Sonnleitner E, Häussler S, Bläsi U. (2016). Sci Rep. 6:39621

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response
Asaoka T, Almagro J, Ehrhardt C, Tsai I, Schleiffer A, Deszcz L, Junttila S, Ringrose L, Mechtler K, Kavirayani A, Gyenesei A, Hofmann K, Duchek P, Rittinger K, Ikeda F. (2016). EMBO Rep. 17(11):1624-1640. Epub 2016 Oct 4.

Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation
Mott AG, Thakur S, Smakowska E, Wang PW, Belkhadir Y, Desveaux D, Guttman DS. (2016). Genome Biol. 17:98.

The Sodium Glucose Cotransporter SGLT1 is an Extremely Efficient Facilitator of Passive Water Transport
Erokhova L, Horner A, Ollinger N, Siligan C, Pohl P. (2016). J Biol Chem epub March 4, 2016

 

The structure and regulation of human muscle alpha-actinin
Ribeiro EA Jr, Pinotsis N, Ghisleni A, Salmazo A, Konarev PV, Kostan J, Sjöblom B, Schreiner C, Polyansky AA, Gkougkoulia EA, Holt MR, Aachmann FL, Zagrović B, Bordignon E, Pirker KF, Svergun DI, Gautel M, Djinović-Carugo K. (2014). Cell 159(6):1447-60.

Structure-Function Analysis of Heterodimer Formation, Oligomerization and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH
Badarau A, Rouha H, Malafa S, Logan DT, Håkansson M, Stulik L, Dolezilkova I, Teubenbacher A, Gross K, Maierhofer B, Weber S, Jägerhofer M, Hoffmann D, Nagy E. (2014). J Biol Chem epub2014 Nov 3.

Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein
Vidilaseris K, Shimanovskaya E, Esson HJ, Morriswood B, Dong G (2014). J Biol Chem 289(34):23870-81.

A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element
Herzog VA, Lempradl A, Trupke J, Okulski H, Altmutter C, Ruge F, Boidol B, Kubicek S, Schmauss G, Aumayr A, Ruf M, Pospisilik A, Dimond A, Senergin HB, Vargas ML, Simon JA, Ringrose L. (2014). Nat Genet 46(9):973-981.

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium
Santos JA, Alonso-García N, Macedo-Ribeiro S, Pereira PJ. (2014). Proc Natl Acad Sci 111(22):E2251-60.

Characterization and Structure of the Vaccinia Virus NF-kB Antangonist A46
Fedosyuk, S., Grishkovskaya I., de Almeida Ribeiro E Jr., Skern T. (2014). J Biol Chem 289(6):3749-62.

Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire
Medvedovic J, Ebert A, Tagoh H, Tamir IM, Schwickert TA, Novatchkova M, Sun Q, Huis In't Veld PJ, Guo C, Yoon HS, Denizot Y, Holwerda SJ, de Laat W, Cogne M, Shi Y, Alt FW, Busslinger M (2013). Immunity 39(2):229-44.

Sufficient amounts of functional HOP2/MND1 complex promote interhomolog DNA repair but are dispensable for intersister DNA repair during meiosis in Arabidopsis
Uanschou C, Roncerat A, Von Harder M, De Muyt A, Vezon D, Pereira L, Chelysheva L, Kobayashi W, Kurumizaka H, Schloegelhofer P, Grelon M (2013).
Plant Cell 25(12):4924-40.