CMP recombinant proteins – University of Copenhagen

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CMP > Facilities/technologies > Recombinant proteins

Recombinant proteins

Mette at a row of PCR thermocyclers 

Expression systems

We have several different heterologous expression platforms at CMP, such as insects (Trichoplusia ni), yeast (Pichia pastoris), ciliates (Tetrahymena thermophila) and bacteria (Escherichia coli, Lactococcus lactis).

Insect cells

In our hands, T. ni cells combined with Baculovirus have proved to be superior when it comes to positive protein tests, quantity and quality of recombinant proteins, and the ability of recombinant proteins to induce antibodies that can surface-label P. falciparum-infected erythrocytes (IEs) and interfere with IE adhesion to vascular host receptors. The recombinant proteins are N-glycosylated by the insect cell, which might be a problem in crystallization studies.

Bacteria

We use E. coli  as a "runner-up" system, as it has enormous benefits over the insect cell system with respect to production parameters (e.g., cheap media, fast growth, numerous genetic variants to chose from, highly developed expression system). However, the recombinant proteins produced in this system are inferior in their ability to induce IE-reactive and IE anti-adhesive antibodies (e.g., Barfod et al. 2006). E. coli-produced recombinant proteins do not get glycosylated, which might be an advantage for crystallization of constructs.

ÄKTAcrossflow

[PICTURE] We use an ÄKTAcrossflow system to purify secreted recombinant proteins. This system allows one-step concentration and dialysis of a given sample, which reduces time and cost of protein production from the insect system.

ÄKTAxpress

[PICTURE] We have three ÄKTAxpress systems at CMP, which are used for the purification of recombinant proteins. The machines are connected to a PC, which is used to design the purification of, monitor, and store the data from each run. The machines uses UV absorbance and conductivity measurements to control washing steps and fractionation of the eluted proteins. They are equipped with several loops making automated multi-step purification procedures possible. Each machine can process up to four purifications in each run, giving us a relatively high protein purification capacity.

Chromatographic methods

Most of our recombinant proteins are hexahistidine- or GST-tagged, making it possible to purify them by affinity chromatography, which is convenient and adequate for most purposes (e.g., animal immunization, antibody purification, ELISA). For more demanding tasks (e.g., crystallization), we use ion-exchange chromatography and gel filtration in order to get proteins of highest purity.