Single‐domain antibody fragments with high conformational stability

Abstract

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, ␤-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T m ‫ס‬ 60-80°C), and display high conformational stabilities (⌬G(H 2 O) ‫ס‬ 30-60 kJ mole −1 ). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.Keywords: Camel heavy-chain antibodies; protein stability; protein folding; circular dichroism; fluorescence; Fourier transform infrared spectroscopy; surface plasmon resonance; high pressure Article and publication are at

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…In spite of their small size, nanobodies bind to their antigens with high affinity (i.e. in the nanomolar range) and specificity [36,37]. In this review, we use the term V H Hs when structural features are concerned, otherwise we use the denomination nanobodies.…”

“…(iii) An additional disulphide bond is present in many V H H sequences, especially those originating from dromedaries; it is generally located between CDR1 and CDR3 (64%) but can also be located between CDR3 and FR2 [39]. In general, V H Hs with longer CDR3 have a higher probability to contain an additional disulphide bridge [37]. This disulphide bond probably restricts the flexibility of long CDRs, which is expected to be entropically counterproductive for binding, and therefore allows a strong interaction [53].…”

“…Nanobodies exhibit a high resistance to temperature (temperature of mid-denaturation, T m , generally comprised between 60 and 80 C), chaotropic reagents (concentration of middenaturation, C m , generally comprised between 2.3 and 3.3 M guanidinium chloride and between 5.8 and 9 M urea) or high pressure (pressure of mid-denaturation comprised between 600 and 750 MPa), and have high conformational stabilities (DG (H 2 O) comprised between 30 and 60 kJ mol À1 ) [37,66]. Consequently, nanobodies can be stored at 4 C for months and at À20 C for years without losing their ability to bind to their antigen.…”

“…The fact that nanobodies exhibit a high stability is especially important for their use as molecular probes to study the process of amyloid fibril formation because denaturing conditions and/or long incubation time (up to several weeks) need generally to be used to trigger the in vitro aggregation of target proteins. Finally, their high stability allows most of nanobodies to be easily concentrated to 1e15 mg mL À1 in standard buffers [46] and to be freeze-dried without losing their functionality [37,69].…”

“…Three nanobodies (named cAb-HuL5, cAb-HuL6 and cAb-HuL22) have been selected by phage display from the blood of a dromedary immunised with the monomeric Wt-HuL; this protein was also used for the panning [37,44,83]. These nanobodies bind to three different epitopes on the surface of lysozyme ( Fig.…”