Eukaryotic Expression Team – University of Copenhagen

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Eukaryotic Expression Team

The Eukaryotic team is  headed by Academic coordinator Giuseppe Cazzamali.

The main focus of the team is to establish a robust eukaryotic expression platform for recombinant target proteins based on two cell systems, HEK293 and Baculovirus-insect cells. HEK293 cells have the potential to produce complex native proteins (such as membrane proteins, glycosylated proteins and protein complexes) otherwise difficult to obtain in other systems. However, protein yields are somewhat lower compared to other expression hosts. Hence, in order to complement the HEK293 system, a Baculovirus-insect cell expression system is currently being implemented.

The main goal for the team is to produce functional proteins. Therefore, full-length constructs are prioritized in both expression systems. This also increases the chance of obtaining proteins with correct folding and relevant post-translation modifications (PTMs). A proper tagging of proteins is very important for the success in the purification process. It has been shown that the STREPII and One STREP tags provide efficient means for purifying proteins produced in the HEK293 system. Protocols are therefore optimized in order to standardize a fast purification procedure for proteins produced in HEK293 cells. C-terminal tag vectors for secretory expression have also been made and proved successful in expressing proteins with their native signal peptide (IGFBPs; HRG; CHI-1). As for the Baculo-insect material, vectors containing both HIS and One STREP tags have been made.

For both HEK293 and Baculovirus-insect cells, we perform a small-scale expression test of all proteins. The large-scale transfection and expression is done in two formats for the HEK293 system; in square bottles (0.5-5L) and shake bags (10L), while the large-scale Baculo-inscet system is performed in shaker flasks (0.5-1L). Baculo high5 cells are the primary choice for large-scale experiments, since they can be grown in suspension.



Fig 1. HEK293 cells transfected with GFP using PEI as transfection reagent. A. Suspension HEK293 cells under inverted contrasting light B. Suspension cells under fluorescent light.

Each target protein undergoes quality control at different stages of expression within the team. At the molecular biology level, each entry clone from the entry clone collection is sequenced before use as templates for PCR cloning. The vector constructs that are made using the entry clones are also 5’ sequenced. For those targets that are selected for scale up experiments, full-length sequencing is also performed. At the protein level, for each small-scale transfection, all target proteins are analyzed by Western blot or SDS-PAGE to evaluate the size of each protein, and after large-scale purification, purity gels are run. 

Recent publications

Wisniewska M, Happonen L, Kahn F, Varjosalo M, Malmström L, Rosenberger G, Karlsson C, Cazzamali G, Pozdnyakova I, Frick IM, Björck L, Streicher W, Malmström J, Wikström M. Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes. J Biol Chem. 2014 Jun 27; 289(26):18175-88.

Larsen T, Yoshimura Y, Voldborg BG, Cazzamali G, Bovin NV, Westerlind U, Palcic MM, Leisner JJ. Human chitotriosidase CHIT1 cross reacts with mammalian-like substrates. FEBS Lett. 2014 Mar 3; 588(5):746-51.

Andresen CA, Smedegaard S, Sylvestersen KB, Svensson C, Iglesias-Gato D, Cazzamali G, Nielsen TK, Nielsen ML, Flores-Morales A. Protein interaction screening for the ankyrin repeats and suppressor of cytokine signaling (SOCS) box (ASB) family identify Asb11 as a novel endoplasmic reticulum resident ubiquitin ligase. J Biol Chem. 2014 Jan 24; 289(4):2043-54.