Table 2.

Recommendations for generating recombinant molecular chaperones with minimal contamination with PAMPs

Before considering the points below, it is important to check that a final recombinant chaperone is properly folded using circular dichroism (CD) or NMR. Improperly folded proteins might lack activity.
1. Express proteins in eukaryotic hosts to avoid suggestions of PAMP contamination.
2. Even if generated in eukaryotic hosts, great care must be taken with protein purification to ensure no bacterial contamination occurs in buffers, piping, columns etc.
3. Always check final preparation of recombinant chaperone with the Limulus assay to ensure that there is no LPS contamination.
4. Check the purity of recombinant chaperones by mass spectrometry as well as SDS-PAGE.
5. If chaperones must be expressed in E. coli, then use the M607 strain, as this has less endotoxic LPS.
6. Chaperones must be treated with polymyxin B to remove LPS and other polymyxin B-binding PAMPs. As some chaperones are very sticky, particularly to polymyxin B, it is best to have the chaperones on some matrix (Ni-NTA, GST etc.). It is better to wash proteins with an excess of polymyxin B than to pass them through a polymyxin B column, as this leads to protein loss.
7. Monitor LPS content in chaperone preparations using the Limulus assay, and decrease LPS content as much as possible.
8. Check that the activity of E. coli expressed proteins is heat sensitive (80–100°C for 10–30 min). Compare with LPS from the strain of E. coli used to express the chaperone.
9. Check that the activity of E. coli expressed proteins is sensitive to proteinase K. Again use E. coli LPS as a control.
10. Check that LPS-neutralising monoclonal antibodies have no effect on the activity of recombinant chaperones.
11. Treat recombinant chaperone with acyloxyacyl hydrolase, which removes 3′acyl groups from LPS and decreases endotoxic activity. Is activity lost?
12. Can the level of LPS contamination in the chaperone preparation account for any of the activity being measured? i.e. if the LPS level is 10 pg/µg chaperone do the cells you are using respond to such levels of LPS?
13. If antibodies to the chaperone are available, use these to immunoprecipitate and remove the chaperone from the potential admixture of moieties being tested or, if antibody is neutralising, add to purified recombinant chaperone and to LPS as a specificity control. If results are positive can these antibodies be used to purify the chaperone?
14. Can chaperone activity be replicated using peptides derived from the chaperone sequence? Do these peptides block the binding of the chaperone to the cell surface?
15. Do the intracellular signalling activities in response to your chaperone and LPS (or other PAMPs) differ? This can be further explored in 16.
16. Use microarray analysis to compare the cellular global transcriptional response to the recombinant chaperone compared to an equivalent activating concentration of LPS.
17. Generate truncation or site-directed mutants of chaperone of interest. If all of the activity of the chaperone is due to contaminants, then this should not influence activity, unless truncation modifies binding to PAMPS. This should be checked.