Question

You suspect there is a protein in blood that has antimicrobial properties. Design an experiment to isolate the protein and test it’s ability to inhibit bacterial growth. How would you determine if it is an antimicrobial peptide or a member of the complement family?

Answer 1

Experiment to isolate the protein:- It is 2 step process

1) Crude extraction:- A crude extract from the blood will be obtained using, the red blood cells were lysed by addition of 0.83% ammonium chloride solution to the blood at a ratio of 3:1. The blood cells were collected by centrifugation (700 × g, 15 min, 4 ◦C), re-suspended in modified phosphate buffer saline (PBSX) buffer (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and sonicated to release the neutrophil granules. These granules will be collected (27 000 × g, 40 min, 4 ◦C), suspended in 10% acetic acid and mixed overnight at 4 ◦C to extract the antimicrobial peptides. The solution containing the peptides will be separated from the granules (27 000 × g, 20 min, 4 ◦C), freeze-dried and dissolved in 0.01% acetic acid.

Peptide purification:- Ion-exchange chromatography will be used to separate the components present in the crude extract, based on the reversible adsorption of the cationic peptides to the immobilized ion-exchange groups of opposite charge. The cationic compounds bound to the anionic resin and the non-cationic molecules were washed through with 25 mM ammonium acetate, then the cationic molecules were eluted with 10% acetic acid.

Experiment to test antimicrobial properties:-

Antimicrobial properties will be determined by the minimum inhibitory concentrations (MICs) of the active pure peptides, the radial diffuse plate assay MIC method was used [20]. The same test organisms as mentioned above were used. The peptide concentrations were calculated using a Protein dotMetricTM 1L Protein Assay (Cat. No. 786-20, 786-21; Geno Technology Inc., St Louis, MO). The size of the clearing on the plate minus the size of the well was plotted against the log of the peptide concentration and a straight line was fitted. The MICs were determined by calculating the point that the line crosses the x-axis. This is the point at which the peptide concentration makes clearing of zero.

Experiment for Peptide characterisation

Mass spectroscopy and N-terminal sequencing of the purified peptides were performed by the Protein Microchemistry Facility, Otago University, Dunedin, New Zealand. Samples for MALDI-TOF-MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry) were prepared by mixing the peptide samples (0.5 L, from 50L total volume HPLC fractions) and the matrix (0.5L of 10 mg/mL -cyano-4-hydroxycinnamic acid in 0.1% TFA, 60% acetronitrile) directly on a stainless-steel slide and leaving them to dry at room temperature. Mass data were collected at near threshold laser fluorescence in the positive ion mode, with a linear instrument (Finnigan LaserMAT 2000; Thermo Bioanalysis, Hemel Hempstead, UK). Where internal calibration was used, this was added to the sample/matrix mixture as indicated. As for electrospray ionisation, the sample (diluted 1:1 from previous dilution in 50% MeCN) was directly infused at 3L/min into the Finnigan LCQ Deca Mass Spectrometer using the Electrospray Ionisation Probe (Thermo Electron Corp., USA). Spray voltage was set to 5 kV, sheath gas (N2) was set to ca. 33 units and the capillary temperature was 220 ◦C. Data were collected over a series of scans and these were averaged to produce the spectrum observed. Automated Edman peptide sequencing was carried out on a glass fibre disk using a Procise 492 Protein Sequencer (Applied Biosystems), with pulsed liquid TFA delivery