Structural biochemistry laboratory at University of South Bohemia in České Budějovice

Laboratoř strukturní biochemie na Jihočeské univerzitě v Českých Budějovicích

Barbora Kascakova, Petra Havlickova, Anna Koutska, Michal Kuty, Ivana Kuta Smatanova^*

Department of Chemistry, Faculty of Science, University of South Bohemia in Ceske Budejovice, 37005, Czech Republic

* Correspondence: ivanaks@seznam.cz; Tel.: +420 387 776 234 (I.K.S.)

The University of South Bohemia (USB) Chemistry Department laboratories are well equipped for a broad spectrum of research. Structural analysis of different viral proteins, enzymes, protein-inhibitor complexes, and large multiple-domain proteins is performed using X-ray crystallography or cryo-EM microscopy. In addition, molecular docking complemented by molecular dynamics is implemented to simulate possible interactions of the tested proteins.

In our laboratory, selected proteins or protein complexes are cloned into vectors suitable for bacterial, baculovirus or mammalian transient protein expression systems. We are currently working with bacterial competent cells: ArticExpress (DE3) Competent Cells, SHuffle® T7 Competent E. coli, BL21-AI™ One Shot™ Chemically Competent E. coli, One Shot™ BL21(DE3)pLysS Chemically Competent E. coli, BL21(DE3) Competent Cells, BL21-CodonPlus (DE3)-RIPL Competent Cells, Rosetta-gami™ 2(DE3) Competent Cells and many more. We also work with the Bac-to-Bac Baculovirus and MultiBac™ expression systems (High Five™, Sf9 or Schneider 2 cell lines). Bhk-21 cells, Hela cell line, Vero cells and others are used for protein expression in mammalian cells. Well-equipped proteomics laboratories with high-capacity orbital shakers (Eppendorf Innova 44r), high-volume centrifuges (Heraeus Multifuge X3R) and ultracentrifuges (Beckman Coulter Optima XPN90) together with well-equipped cell culture and BSL2 facilities are suitable for expressing proteins at sufficient concentrations for further purification.

Two AKTA pure 25 chromatography systems (room temperature or 4 °C conditions) are used to purify the target proteins. Many commercial chromatography columns are available that are suitable for both systems. In addition, the room temperature size exclusion chromatography system is complemented by Dawn Heleos II and Otilab T-rEX detectors, which are used for accurate analysis of purified samples by measuring multi-angle static light scattering (MALS) and refractive index. The most commonly used techniques in the proteomics laboratory are affinity chromatography, anion exchange chromatography, heparin chromatography, and SEC-MALS of small globular proteins to large macromolecular complexes.

Structural analysis of proteins is performed by basic X-ray crystallography. Standard crystallization techniques (vapor diffusion, microdialysis and free-interface diffusion) and advanced techniques (counter diffusion, capillary and in gel crystallization,) are used to prepare diffracting quality crystals. In case of problematic crystallization, seeding, co-crystallization with ligands or crystallization in living cells are used. Before each crystallization experiment, a dynamic light scattering instrument (unless SEC-MALS is used) is implemented to confirm the homogeneity of the molecules. The Oryx Nano (and soon Oryx 8) crystallization robotic system and several incubators with different temperatures are at disposal to screen crystallization conditions to ensure that the best possible conditions for crystal growth are found. Diffraction measurements are performed on synchrotron-based macromolecular beamlines at BESSY (Berlin, Germany), DESY (Hamburg, Germany) or ESRF (Grenoble, France) according to standard application procedures. Data processing and further structure solution steps are performed on computational hardware and software located in the Structural Biochemistry Laboratory and operated by trained personnel. A new crystallographic method, crystallization in living cells, is beginning to be incorporated into the methods used in our laboratory.

Figure 1

These researches are supported by European Regional Development Fund-Project, MEYS (No. CZ.02.1.01/0.0/0.0/15_003/0000441); by the Grant Agency of the Czech Republic (Grant No. 19-14704Y) and by the Grant Agency of the University of South Bohemia (grant No. 04-046/2022/P).