Purification and Identification of a Functionally Defined Molecule

In the search for a causative agent responsible for a specific bioactivity, in our case histamine release, the general approach is to remove the maximum amount of overall protein, while maintaining the function of interest by sequential purification steps. Our strategy involved monitoring total protein by absorbance readings (A280) and function by histamine-release assays using basophils from our subpopulation of allergic donors. The purification steps were: (1) repetitive column chromatography; followed by (2) gel electrophoresis.

3.1.1. Repetitive Column Chromatography

1. Culture the U937 cell line to obtain 50 L of supernatant containing histamine release activity.

2. Concentrate the supernatant to 14 mL and apply to a Sephadex G75 gel filtration column (5 x 90 cm) with a bed volume of 1.8 L in 0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4.

3. Collect 140-13-mL column fractions and monitor the protein concentration by A280 and the bioactivity by histamine release.

Histamine Release

Fig.1. Example of column chromatography using size-exclusion Sephadex 675 is shown. The protein tracing (•) is demonstrated to be predominately separated from the biologic activity (O, histamine release). Fractions were pooled (tubes 72-100) to maximize biologic activity and minimize protein contamination.

Fig.1. Example of column chromatography using size-exclusion Sephadex 675 is shown. The protein tracing (•) is demonstrated to be predominately separated from the biologic activity (O, histamine release). Fractions were pooled (tubes 72-100) to maximize biologic activity and minimize protein contamination.

4. The histamine release assay is routinely used in our laboratory (4), but any functional measure that is of interest can be used to track the purification, for example, with a commercially available ELISA.

5. Figure 1 shows the graph of these chromatography parameters and the fractions chosen to be further purified. This is a typical graph used to determine the column fractions of interest based on retaining maximal function and minimal protein.

6. Pool, concentrate, and dialyze the bioactive fractions into the MONO Q CB.

7. Perform several chromatographic runs on a MONO Q anion exchange column in 0.2 M Tris-HCl, pH 8.0 with an increasing salt gradient from 0 to 1 M NaCl.

8. Collect 30-2-mL column fractions and monitor the fractions as described above.

9. Pool the bioactive fractions from all of the MONO Q runs, concentrate, and dialyze against the Superdex CB.

10. Run numerous Superdex columns in 1X PIPES and collect 65-2-mL column fractions.

11. Monitor as previously described and pool and concentrate the active fractions for gel electrophoresis (see Note 1).

3.1.2. Gel Electrophoresis

1. Electrophoris the concentrated column fractions on SDS-PAGE using standard methods.

2. Blot onto a polyvinylidene difluoride membrane and stain with Coomassie blue.

3. Excise the visible bands from the membrane and send to an institutional core or a commercial laboratory for amino-terminal sequencing.

4. Once the sequence is obtained, online resources such as the NCBI Entrez page (http://www.ncbi.nlm.nih.gov) are useful for determining if the sequence matches a known sequence in a data base. In our case, a search matched one sequence to a protein in the data base with no known function called translationally controlled tumor protein (accession no. X16064, Swiss-Prot no. P13693).

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