Protein expression

Expression of recombinant proteins

Protein expression laboratories that integrate with a central recombinant protein expression core facility, at present being established at the University of Aarhus. This combination provides us with enhanced access to large-scale fermentors, an increased number of running expression systems, and numerous efficient down-stream services. Our main expression systems include E. coli and Pichia pastoris (yeast) where isotope labeling will be conducted in minimal medium. The choice of system depends on several factors including size of the protein of interest, type of post translational modifications and need for 13C and/or 15N labelling.
Mammalian expression systems is used mainly for production of recombinant proteins for biochemical and biophysical investigations. We will have strong focus on different pET vector protein based methods and on ligation of labeled and non-labeled fragments using intein-based strategies.

Proteins are obtained primarily in two separate ways:
• Purified from a natural source.
• Recombinant material.

Protein expression Laboratory

Cell culture facility

Our state-of-the-art, self-contained cell culture facility houses all equipment needed for the growth, maintenance, and analysis of cells. It is used for the expression of recombinant protein and the proliferation of mammalian cells.

Protein purification

Hydrogen/deuterium exchange mass spectrometry

In recent years, the field of mass spectrometry have seen the introduction of new techniques, developed to supplement the existing NMR and X-ray crystallography procedures used during structural analysis of proteins. One of these novel techniques is hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange mass spectrometry takes advantage of surface exposed hydrogen’s (H) ability to exchange with hydrogens in the environment. When placed in high deuterium content (D2O), surface hydrogens of a protein will exchange to deuterons (D). This is associated with an increase in mass, which can be measured by mass spectrometry.

Hydrogen/deuterium exchange mass spectrometry exploits the variation of the exchange ratio of the backbone hydrogen, depending on the exposure to a solvent. By dissolving the protein in deuterated water and leaving the exchange run for different time periods, it is possible to identify which part of the protein is buried in the structure, taking part in a binding site or exposed to the solvent.

Hydrogen/deuterium exchange mass spectrometry of glucagon. Monomeric (middle spectra) or fibrillated (upper spectra) glucagon was diluted into D2O. The exchange was quenched after 10 minutes. The samples were analyzed by MALDI-MS. Monomeric glucagon shows a significant exchange as seen by a m/z shift of approximately 15, indicating a relatively unprotected structure. Upon fibrillation, the glucagon peptide becomes highly protected and shows only an average exchange of two backbone amides. All exchanges are relative to a unexchanged monomeric glucagon sample (lower spectra).

Hydrogen/deuterium exchange mass spectrometry does not give the same structural resolution as NMR and X-ray crystallography, however the method has several advantages. Hydrogen/deuterium exchange mass spectrometry is much more sensitive, significantly lowering the amount of material needed for a study. Sample preparation and analysis are considerably faster than for NMR and X-ray crystallography. Additionally, samples of unlimited size and complexity as well as relatively impure samples can be analyzed by hydrogen/deuterium exchange mass spectrometry. Furthermore, hydrogen/deuterium exchange mass spectrometry can handle insoluble samples such as fibrils or components in the extra-cellular matrix.

Mass spectrometry and N-terminal sequencing

The laboratory hosts a protein-analyses core facility offering LC-MS/MS-based protein identification and N-terminal sequencing at favourable prices for researchers at Aarhus University (for more information see services).

Currently our mass spectrometry platform consists of :
1. Micromass/Waters Q-Tof Ultima Global mass spectrometer used for matrix assisted laser desorption/ionization (MALDI) analyses, and 
2. Micromass/Waters Q-Tof Ultima API mass spectrometer connected in-line to an EASY-nLC nano HPLC system from Proxeon Biosystems. 
The later is used for LC-MS/MS-based protein identifications.

Prior to identification, the protein of interest is typically subjected to gel-electrophoresis, followed by in-gel digestion and micro-purification of the resulting tryptic peptides. Once the sample has been analyzes, the data is processed using Masslynx software from Micromass/Waters and the Mascot search engine from Matrix Science to query relevant protein data-bases.

N-terminal sequencing is performed using automated Edman degradation on a PROCISE® sequencer, from Applied Biosystems, with on-line phenylthiohydantion-derivative HPLC analysis on a 140C Microgradient System from Applied Biosystems.

Mass spectrometry laboratory
Micromass Q-TOF Ultima API: A hybrid quadrupole time of flight mass spectrometer fitted with a electrospray source
Micromass Q-Tof Ultima Global: A hybrid mass spectrometer whith both (nano)electrospray and MALDI interface
Applied Biosystems PROCISE™ 494 HT: Protein Sequencer containing 4 reaction cartridges for high throughput automated Edman degradation
Nano LC MS/MS inlet

Protein and peptide purification

Protein and peptide purification comprises a series of processes intended to isolate a single type of protein/peptide from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. We routinely purify proteins from natural sources as well as recombinant proteins. Our starting material is biological tissue such as plasma (human), cornea (porcine), skin (bovine) and aorta (bovine) or media from cell cultures.

The various steps in the purification process involve extraction, differential precipitation and separation by several chromatographic methods. Separation based on the principles of chromatography exploit differences in protein size, physical-chemical properties and binding affinity. Our laboratory is equipped to handle purification of proteins and peptides in the range of grams to nano-grams. We routinely use:
• Size exclusion chromatography
• Reverse phase chromatography
• Ion exchange chromatography
• Affinity chromatography
   - Metal binding
   - Heparin binding
   - Immunoaffinity chromatography
   - Purification of a tagged protein


ÄTKA™ purifier: The GE Healthcare ÄKTA purifier is a high end liquid chromatography system designed for fast and reliable separations of both proteins and peptides
ÄTKA™ explorer 10: Chromatography system developed by Amersham Biosciences, compatible with a wide range of purification techniques for both proteins or peptides
ÄTKA™ prime plus: System supplied by Ammersham Biosciences with pre-programmed templates for simple and fast purification of proteins at laboratory scale


Protein chemistry lab 2009
The main laboratory

Zuzana Valnickova
Laboratory Coordinator
Laboratory for Proteome Analysis and Protein Characterization
Department of Molecular Biology
Aarhus University
Gustav Wieds Vej 10C
8000 Aarhus C

Telephone: 89425005