Purification of CD2

 

1) Cells are pelleted (~3 - 4000 g for 10 min.) and resuspended in pre-chilled 50 mM malonate (pH 5.2-5.3) with 1mM EDTA (~40ml of buffer per litre of culture).

2) Lyse the cells with French press.

3) Pellet the cell debris (~20000g for 30 min.). Carefully transfer the supernatant to a new tube. Check pH (5.4~5.5).

4) Equilibrate the SP-sepharose column with the malonate buffer (~1- 1.5 hour at flow rate of 2 ml/min). Load the crude cell lysate onto SP column, wash and then elute using a salt concentration gradient on the FPLC (programme 5).

5) Analyse the eluate with SDS-PAGE and pool the desired fractions.

6) Equilibrate the gel-filtration column with 20 mM phosphate (pH 5.4) for 2 hours at 1-1.5 ml/min. Load the pooled fractions onto the gel-filtration column (load ~ 5 ml per run). This should desalt and buffer-exchange the CD2 protein as well as removing the larger contaminants.

7) Analyse the fractions collected. If necessary, load the pooled fractions onto the S-10 cation exchange column and elute using a stepwise pH elution.

9) Concentrate protein using Centriprep (~1000g) to a final concentration of 1-2 mM. The protein has a tendency to precipitate at high concentration (especially between pH 5 to 7), therefore take care to concentrate slowly. Expect protein yield of ~20-40 mg/L if there is little precipitation. The protein thus obtained is pure enough for NMR studies but protease inhibitors (PMSF + EDTA) need to be added (It is preferable that all buffers used in the purification should contain 0.1 mM PMSF and 0.5 mM EDTA).

Note:
i) CD2 should be purified as quickly as possible to avoid the problem of proteolysis. Do not stored sample in fridge for more than a day between purification steps, stored at -20 °C instead.
ii) Avoid as many steps as possible when purifying protein. Proteins are lost during each purification step, therefore fewer steps minimise lost of proteins.
iii) Gel filtration (size exclusion) is often used as the final step in protein purification but here it is used to desalt and buffer exchange the protein (therefore no need for dialysis) as well as removing the contaminating proteins of larger sizes. If the CD2 appeared purified, no further steps are necessary, otherwise purify the protein further using a mono-S cation exchange column (elute at pH 6.5, 7.5 and 8.5 - CD2 should elute at pH 7.5) or other columns such as hydrophobic interaction column.
iv) For SP and Mono-S columns after the elution of the protein is completed, wash the column with 1 - 2 M NaCl solution to remove any bound proteins. Wash again with low salt buffer and then store the column in 20% ethanol.
v) The SP colomn in first step in the purification may be replaced with S-sepharose. This give a sharper peak and may need only a further gel-filtration step.
vi) The elution of the protein is monitored with the OD at 280 nm which is the absorption maximum of tryptophan (the main component) and tyrosine (which absorb less strongly than trp).
vii) The pH of the crude cell lysate is kept at 5.3 to ensure that the protein will bind well to the SP column but not so low as to precipitate most of the E.coli proteins as well as CD2. Most E. coli proteins have isoelectric point <5 therefore tend to precipitate out at low pH - this would be useful for removing most of unwanted bacterial protein but not if your protein precipitate out as well. It is, however, useful to note that results indicate that maximal dimer formation of CD2 (and which appeared to be associated with the precipitation of CD2) occurs at ~ pH 6 (actually probably pH 5.7) while at pH < 5, monomer predominates. It may therefore be possible to start a lower pH (say ~4 -4.5), but there will be considerable precipitation of bacterial proteins which may carry the risk of co-precipitating CD2 as well.
viii) With precipitation the main problems in CD2 purification, it may be worth considering using the His-tag construct which is purified at a higher pH where less precipitation is observed. All the wild-type and mutant CD2 are constructed in both the native and his-tag versions. This is especially useful for some mutants which precipitate heavily in acidic condition (e.g. K43A, L38A, Y81A?) using the normal method of purification. Therefore use the His-tag purification method for the his-tagged version of CD2 (note that there shouldn't be any EDTA in the buffer in this first step of purification). Add ~1-5 mg of carboxypeptidase after the His-tag purification step and incubate if you wish to remove (one or two histine residues, however, will remain). This is followed by a gel-filtration step. If you are purifying the mutant proteins that may precipitate heavily in acidic pH, then it is advisable not to use the S-10 column next. The first 2 steps are normally sufficient to get a reasonably pure protein if the his-tag step is done carefully. However, if the protein sample still look impure on the gel, load the His-tagged protein (the his-tag should not be cleaved for this and may not work for some of the mutants) onto the S-10 column at pH 6.5 (there may not be so much precipitation at this pH), then wash with pH7.5, and elute at pH 8.5.