Bio Tools

 

The main assignment of BioLab is to produce labelled membrane proteins and membrane-active peptides of our interest for NMR-analysis as well as to carry out bioassays for characterisation of antimicrobial and haemolytic activity of chemically synthesized by peptide synthesis 3D-, 15N- and 19F-labelled peptide derivatives. How peptide penetrate and alter the cell membrane we observe using fluorescence microscopy and electron microscopy in collaboration with LEM, KIT.

Expression of membrane proteins

For production of membrane proteins we use the advantages of molecular cloning with following expression of recombinant 13C/15N- and 19F-labelled membrane proteins in E.coli systems. Membrane proteins of our interest are TatA, TatC, E5, PDGFß-receptor and Mslc.

Gramicidin S production
Especial effort we undertake to production and study of antimicrobial peptide Gramicidin S and its producer Aneurinibacillus migulanus (former Bacillus brevis). Today’s state of producer strains ATCC 9999T=DSM 2895T, DSM 5759 and DSM 5668 in culture collection does not allow to produce Gramicidin S with a high yield or at all, due to dissociation of producing rough (R) colony phenotypes into non-producing smooth (S) colony phenotypes. We have found conditions for reversible dissociation of S phenotypes into the R phenotypes to save industrially valuable properties. To elucidate molecular structure of Gramicidin S and its interaction with model and bacterial membrane we develop optimal growth conditions for production of 13C/15N labelled peptide in minimal medium. Other point of our interest is looking for a possible role of Gramicidin S in producer cells to understand - how could it be possible for cells accumulate 250mg of Gramicidin S pro 1g dry cell weight and which cell structures could contain such a huge amount of peptide. We use fluorescence and electron microscopy to compare ultrastructure of producing and non-producing cells.
Bioassays for studying of antibacterial and haemolytic activity of peptides

 Gramicidin S, which is selective against Gram-positive bacteria and Polymyxin B, which is selective against Gram-negative bacteria are in our BioLab a standard, model peptides. These peptide allow us to be sure in results by checking antimicrobial activity of chemically synthesized labelled peptides in follows methods:

 

  • Agar diffusion method (screening)
  • Microdilution method (modified MIC - minimal inhibitory concentration assay)
  • Synergy testing (checkerboard assay)
  • alamarBlue® test (inhibitory of bacterial respiration)
  • LIVE/DEAD® BacLightTM bacterial viability test (see fluorescence microscopy)

 

Due to antimicrobial peptides are not only able to destabilize bacterial membrane, but also membrane of red blood cells, we characterize their activity against this cells in haemolytic assay with human erythrocytes. Gramicidin S shows a high haemolytic activity (sample movie) and we use this peptide in experiments as a positive control. Haemoglobin release and appearance of erythrocyte ghost after 2 min exposure with 20 µg Gramicidin S/ml could be observed live in Axioscop 40 (Carl Zeiss) under phase contrast with Objectiv “A-Plan” 100x / oil Ph3. For determination of minimal haemolytic concentration, for example, 50% haemolytic activity of peptides (HC50),  we measure haemoglobin release of final erythrocyte concentration 0.5% at 420 nm (red points) or at 540 nm (blue points) in comparison to entirely haemolysis (HC100),  which we obtain by addition of 0.1% Triton X-100. We plan also to study cytotoxicity of antimicrobial and cell penetrating peptides on some human cell cultures.

 

 

 

 

 

 

 

 

Fluorescence microscopy
Fluorescent staining of bacterial and human cells we use for different tasks. For example, we study bacterial cells treated with peptide antibiotics to observe their antibacterial affect on the whole bacterial population and to compare fluorescence results with MIC values. DEAD/LIVE BacLightTM staining of E.coli DSM 1103 treated with 20 µg/ml PGLa reveals red fluorescence of some damaged cells (A). Due to the presence of ornitine in Gramicidin S it is possible to carry out pH selective staining of this peptide with 5(6)-carboxy-fluorescein-hydroxysuccinimide, which allows us to estimate Gramicidin S amount in producer cells and to distinguish producing and non-producing phenotypes (B). Penetration of peptides into the HeLa Cells (green) incubated with cell penetrating peptides BP100 (C) and HIV-TAT (D) shows differences in peptide uptake.  BP100 is taken up via endocytosis, what results in a punctuated pattern. In contrast HIV-TAT is bound onto membrane (red) and distributed uniform within the cell (yellow colour results as a mixture of red and green fluorescence).