Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains.
BACKGROUND
Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein.


RESULTS
Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature.


CONCLUSIONS
Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins.
Olichon
Aurelien
A
Cell Biology Unit, EMBL--Meyerhofstr. 1, D-69117, Heidelberg, Germany. aurelien.olichon@embl.de
Schweizer
Daniel
D
Muyldermans
Serge
S
de Marco
Ario
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D002161:Camelids, New World; D005591:Chemical Fractionation / Q000379:methods*; D006358:Hot Temperature; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal