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
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.
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
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
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
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.
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: email@example.com. Further details
can be found at http://proteinfacility.nki.nl.
The polybasic insertion in autotaxin α confers specific
binding to heparin and cell surface heparan sulfate
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,
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
J. Struct. Biol. (2012) 179, 46-55.
Radner S, Celie PH, Fuchs K, Sieghart W, Sixma TK, Stornaiuolo
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,
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
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
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,