Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains

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. [8]. 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.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.There has been an increased interest in antibodies in recent years, both because of their clinical applications and their use in basic research [1]. Conventional antibodies (mono- and poly-clonal) present several shortcomings, such as their bulky structure, tedious and expensive preparation, limited opportunities to introduce mutations, and the immunogenic response they can induce when used in therapy. For these reasons, a techniques aimed at recombinant expression of antibodies selected from both immunized and na？ve/synthetic libraries can be a convenient alternative [2].The most common format for antibody recombinant expression is probably the single chain antibody (scFv), in which the heavy and light variable regions are linked together. Polybodies, with higher avidity for the antigen than single scFv molecules, can be obtained by varying the length of the linker or by connecting via a flexible hinge to an amphipathic helix [3,4]. ScFvs have been widely used to identify antigen