Question

From Hung et al., (2017) “Purification,characterization and biological effect of lectin from the marine sponge Stylissa flexibilis (Lévi, 1961)” Specimens were cut into small pieces, ground with a mixer machine and extracted (1:2, w/v) with 50 mM Tris–HCl, 150 mM NaCl buffer (pH 7.5) for 6 h at 4 °C. After filtration through a cheese cloth, the filtrate was centrifuged at 3500 × g for 30 min at 4 °C. To the supernatant, cold absolute ethanol (− 20 °C) was added to attain a final concentration of 80% and the mixture was kept at 4 °C overnight. The resulting precipitates were collected by centrifugation at 3500 × g for 30 min at 4 °C. The pellet was washed three times by cold absolute ethanol (− 20 °C) and centrifuged at 3500 × g for 30 min at 4 °C. The pellet was thoroughly dialyzed against 20 mM Tris–HCl buffer (TB), pH 7.5. The non-dialyzable fraction was applied to a DEAE Sepharose fast flow ion exchange chromatographic column (1.6 × 20 cm), equilibrated with the above buffer. Unbound proteins and pigments were eluted with above buffer at a flow rate of 10.0 mL min− 1 until the column effluents showed absorbance of less than 0.002 at 280 nm, lectin was eluted with 0.5 M NaCl in 20 mM Tris–HCl buffer, pH 7.5; the active fractions were pooled, concentrated by ultrafiltration, and dialyzed against 50 mM Tris–HCl, 150 mM NaCl buffer (pH 7.5). The concentrate was subjected to gel filtration on a Sephacryl S-200 column (1.6 × 60 cm) equilibrated with above buffer. The column was eluted with the same buffer at a flow rate of 0.8 mL min− 1 and the active fractions were collected. A. Outline the steps in this purification from specimen to purified protein. Describe how each step works. (If a column is run, describe how the column separates proteins.) B. Explain what the DEAE Sepharose column tells us about lectin. C. What technique could be used to verify the lectin was in the precipitate that formed after ethanol was added? D. The sample was dialyzed to remove what?

Answer 1

Lectins are proteins that bind to the carbohydrates molecules.

As marine sponges grows in water so their surface contain hydrophocbic coating and cell membrane contain lipids and other molecules.

1. Outline the steps in this purification from specimen to purified protein. Describe how each step works. (If a column is run, describe how the column separates proteins.)

In first grinding of samples in buffer maintain the pH of isolated sponges tissue and proteins. Grinding will rupture the cells and cell content will come into the stable buffer.

Filtration will remove the big insoluble particles from the mixture. Small insoluble particles will remove by cetrigugation.

Washing with alcohol dissolve the lipid components of the sponge and cells and denature the cell membrane proteins along with the lectins. Repeated washing precipate the proteins and remove the lipid substances from the sample.

Dialysis will remove the absolute alcohol and re-nature the proteins along with lectins.

Now this fraction applied to DEAE column which is positively charged hence negatively charged residues and proteins will bind to the DEAE beads of the column. Washing step will remove the unbound proteins and UV will drop at this step. By applying NaCl will remove the bound proteins and lactins.

Active fraction will bring to the same ionic concentration in which buffer gel filtration chromatography will performed. In gel filtration chromatography only 1% sample is loaded therefore sample will be concentrated. In gel filtration active fractions will be collected.

B. DEAE beads are positively charged beads hence only negatively charged proteins or which are high –COOH group containing amino acids such as aspartate and glutamate rich proteins are present in lectins.

C. One way to find out that the non-precipitated fraction also tested for the lectin specific test that suggests that after ethanol ppt major amount move to which fraction. This is knwn as specific activity.

D. After ethanol wash sample is washed to remove the ethanol that bound to protein and causing denatuation. After dialysis excess of salt removed by the dialysis because fraction eluted at 0.5 M NaCl and gel filtration required 150 mM and 50mM tris Cl.