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Protein facility

The Protein facility of the Netherlands Cancer Institute started its activities in 2008 and shares lab space and equipment with the structural biology groups of Anastassis Perrakis and Titia Sixma at the Division of Biochemistry (B8). The facility offers an excellent infrastructure for biochemical, biophysical and structural biology experiments and contributes to a variety of research projects. Projects can be submitted to the facility (see contact details below) and will be discussed in terms of costs and feasibility before initiation of the project. Projects are classified as short-term (less than 3 days of work) and long-term (more than 3 days of work). For long-term projects hands-on assistance from the applicant is required. The activities of the facility can be subdivided into different categories.

Construct design and cloning

Expression of active and functional protein is dependent on many different factors. The facility can assist in the design of selective protein fragments using structural- and bio-informatics tools (see http://xtal.nki.nl/ccd/ ) to increase the chance of obtaining soluble and active protein. A variety of LIC (Ligation independent Cloning) expression vectors is available to facilitate expression of proteins fused to different purification and solubility tags. Cloning has to be performed by the applicant.

Protein production

Over the years, many proteins have been expressed and purified for a variety of applications, including structural studies, pull-down experiments, binding studies, immunization to obtain antibodies, enzymatic assays and other biochemical applications. The facility uses different organisms for over-expression of recombinant proteins. The most commonly used host is the bacterium Escherichia coli. Expression trials can be performed at 3 ml scale and can be scaled up to production levels of 6 to 12 liter cultures to obtain milligram quantities of protein. Big proteins and/or multi-domain eukaryotic proteins however, are often difficult to express in E. coli. In that case, proteins can be produced in Sf9 insect cells using the Baculovirus system. Up to 4 liters of insect cell culture can be used for the production of a single batch of protein. The production of extracellular proteins can be performed in different Human Embryonic Kidney (HEK293) cell-lines. Both transient expression as well as expression from stable cell lines are supported by the facility. Large-scale expression can be performed in roller-bottles that are placed in a dedicated incubator with a capacity of 90 roller-bottles.

Protein purification

A first step in protein purification is often affinity-based using resin that specifically interacts with (the tag of) the protein. One Äkta Micro an four Äkta FPLC system are available for automated protein purification chromatography at analytical scale (0.1 to 1 milligram of protein) and large scale (milligrams of protein), respectively. Each system is equipped with a series of size exclusion and ion-exchange columns and can also accommodate other columns.

Antibody production

A large number of hybridoma cell-lines, each expressing a different monoclonal antibodiy, have been collected at the Netherlands Cancer Institute. The facility provides a service for the production and purification of antibodies upon request of our researchers. A list of hybridoma cell-lines and antibodies is available.

Biophysical characterization

The facility offers a variety of biophysical techniques to characterize protein properties and function, including protein stability, protein complex formation, oligomerisation and protein interactions. Protein thermal stability can be analyzed in different buffers and/or chemical conditions using a Thermal Shift Assay (also known as Thermofluor) to identify optimal protein purification- and storage conditions. A multi-angle laser light system (MALLS) coupled to size exclusion chromatography is available to measure the molecular weight of a protein (-complex) which is helpful to determine stoichiometry and oligomerisation status. A number of different systems is available to examine the interaction between proteins and (macro-) molecules like DNA, peptides, proteins and small organic compounds. These instruments include a Biacore T100 instrument for surface plasmon resonance; a VP-ITC to measure interaction using Isothermal Titration Calorimetry, a fluorescence plate reader (e.g. for fluorescence polarization assays) and a stopped-flow system (for measuring fast kinetics).

Crystallization screening

The facility has access to an automated high-throughput crystallization screening platform. Crystallization conditions can be screened for in 96-well format in 200 nanoliter droplets at 4 °C and 20 °C. Plates are prepared by (nanoliter) liquid handling robots and crystallization trials are visualized over time by automated imaging systems that are accessible via web-servers.

Mass spectrometry

A LTQ-Orbitrap Discovery mass spectrometer coupled to a nano-liquid chromatography (nano-LC) system is used for protein identification purposes. Proteins (in solution or on SDS-PAAGE gel) are digested with trypsin and proteolytic peptides are analyzed using peptide mass fingerprinting.

Protein facility staff and support

The protein facility has a dedicated staff maintaining the facility, providing expert advice, access to expression systems and hands-on support. Information about the facility and/or requests for projects can be send to Patrick Celie: p.celie@nki.nl. Further details can be found at http://proteinfacility.nki.nl.

Publications

The polybasic insertion in autotaxin α confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
J. Biol. Chem. (2013) 288, 510-9.
Houben AJ, van Wijk XM, van Meeteren LA, van Zeijl L, van de Westerlo EM, Hausmann J, Fish A, Perrakis A, van Kuppevelt TH, Moolenaar WH.

Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding.
J. Am. Chem. Soc. (2012) 134, 13357-65.
Heidebrecht T, Fish A, von Castelmur E, Johnson KA, Zaccai G, Borst P, Perrakis A.

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells.
J. Struct. Biol. (2012) 179, 46-55.
Radner S, Celie PH, Fuchs K, Sieghart W, Sixma TK, Stornaiuolo M.

Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2011) 67, 1545-7.
Singh KA, Jagannadham MV, Rao GR, Celie PH.

Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state.
FEBS Lett. (2011) 585, 3593-9.
Farenc C, Celie PH, Tensen CP, de Esch IJ, Siegal G.

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases.
J. Struct. Biol. (2011) 175, 113-9.
Luna-Vargas MP, Christodoulou E, Alfieri A, van Dijk WJ, Stadnik M, Hibbert RG, Sahtoe DD, Clerici M, Marco VD, Littler D, Celie PH, Sixma TK, Perrakis A.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.
J Struct Biol. (2011) 175, 159-70. Busso D, Peleg Y, Heidebrecht T, Romier C, Jacobovitch Y, Dantes A, Salim L, Troesch E, Schuetz A, Heinemann U, Folkers GE, Geerlof A, Wilmanns M, Polewacz A, Quedenau C, Büssow K, Adamson R, Blagova E, Walton J, Cartwright JL, Bird LE, Owens RJ, Berrow NS, Wilson KS, Sussman JL, Perrakis A, Celie PH.

Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation.
EMBO Rep. (2011) 12, 342-9.
Maspero E, Mari S, Valentini E, Musacchio A, Fish A, Pasqualato S, Polo S.

People working at the Protein facility

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Patrick Celie

Protein facility manager

Personal details

Experience

Responsible for the day-to-day operation of the facility. Expertise:  Molecular biology (LIC cloning); Protein production (E. coli, baculovirus, sf9 insect cells, mammalian cells) and purification; Isothermal Titration Calorimetry (ITC); (high-throughput) protein crystallisation.

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Magda Stadnik

Technician

Personal details

Experience

After graduation at the Warsaw University of Technology, MSc in Biotechnology, I started my internship in the laboratory of Anastassis Perrakis in 2010. After 3 months training at the division of Biochemistry I continued my career as Research Analyst in the Protein Facility. I am responsible for protein production and purification, crystallization and biophysical characterization. Since 2015 I am part of the iNEXT Project Management team, taking care of the communication to facility users of all involved European participating facilities, managing all users aspects with respect to their access to the Netherlands Cancer Institute services, providing technical support, document and budget management.

 

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Alex Fish

Postdoctoral Fellow

Experience

Major scientific project DNA mismatch repair.

Major scientific expertise biophysical characterization of bimolecular interactions using different techniques such as SPR, ITC, MALLS and spectroscopic methods.

Experience in protein purification, X-ray crystallography, protein mass spectrometry.

Operator of biophysical instruments and mass spectrometer. Provides support for biophysical experiments. Expertise: Surface Plasmon Resonance (SPR); Fluorescence Polarisation (FP); Isothermal Titration Calorimetry (ITC); mass spectrometry.

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John de Widt

Technician

Experience

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