Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase

Abstract

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N 2 O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies TM ). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

Viral infection modulation and neutralization by camelid nanobodies

“…Lymphocytes were isolated from blood samples of an immunized llama, and a nanobody phage display library was generated (28). Specific nanobodies were selected by panning (29).…”

“…Nine BP TP901-1 -specific binders were selected and sequenced (Fig. S1) before being subcloned into the pHEN6 expression vector (28). For the Tal N terminus, we obtained four different nanobodies, of which two could be expressed and thus, were further characterized (Fig.…”

“…A llama (Llama glama) was immunized with the purified BP TP901-1 and Tal N-terminal domain (encompassing residues 1-386) (Capralogics INC). Lymphocytes were isolated from blood samples, and a nanobody phage display library was generated using standard procedures (28). The nanobody library contained about 3 × 10 8 independent transformants, of which 95% of the clones harbored an insert of expected nanobody gene size as determined by PCR on individual colonies.…”

“…The sequence analysis of positive clones showed nine and four different clones, respectively, being subcloned into pHEN6 expression vector. Nanobodies expression and purification were performed as described (28).…”

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines

“…The VHHs are famed for a good expression yield and an excellent solubility compared to most mice or human two chains-derived antibody fragments [review in 47]. They may be used in a range of assays [48], for protein purification [49], and as an aid to crystallization [50]. Multiple therapeutic and diagnostic agents have been or are being developed from VHHs [51,52].…”