1996). the stability of the resulting structure and its affinity for lysozyme. The engineered NCS-CDR3 presents a structure comparable to that of the wild-type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements exhibited that the new NCS-CDR3 specifically bound lysozyme. Keywords: neocarzinostatin, CDR3 loop, camel antilysozyme, drug targeting The development of soluble proteins that recognize given target molecules (ranging from small chemical compounds to macromolecular structures) Ebselen is usually of increasing importance in therapy and biotechnology. In the past decades work in this direction was centered mainly around the engineering of antibodies as they display specific and tight binding to a huge variety of molecular compounds. However, antibodies and their functional fragments present several disadvantages from a practical point of view. First, they are large molecules: Even the smallest antigen-binding fragment, Fv, consists of approximately 250 amino acids. Second, they are composed of two different polypeptide chains, necessitating complicated Ebselen cloning actions for the pair of genes, sometimes resulting in unstable domain association in the case of Fv (Hoogenboom 1997). A single-chain engineered fragment of immunoglobulin called ScFv consists of the VH and VL subunits joined by a synthetic peptide (Bird et al. 1988). It is the most widely used fragment of immunoglobulin in biotechnology because it can be produced in various hosts and because it is usually relatively stable (Harris 1999; Hudson 1999; Kousparou et al. 2002). However, in many cases, ScFv can only be produced in small quantities. To overcome the drawbacks of the immunoglobulins, other protein scaffolds have been developed to create new types of binding protein. The proteins used include protease inhibitors, DNA-binding protein, cytochrome b562, helix-bundle proteins, disulfide-bridged peptides, lipocalins, and their derived anticalins (Nygren and Uhlen 1997; Skerra 2000). Several approaches have been used to obtain new ligand-binding functions drawing on either rational design experiments in conjunction with site-directed mutagenesis (Riechman et al. 1988; Essen and Skerra 1994; Schiweck and Skerra 1995; Ellis et al. 1996; van den Beucken et al. 2001), or combinatorial molecular biology methods (Smith Rabbit polyclonal to AGAP 1991; Wells and Lowman 1992; Hoess 1993). Here, with the same goal, we have explored a new scaffold, the neocarzinostatin, as a potential support for new conversation capacities. Neocarzinostatin belongs to the family of bacterial chromoproteins. The known members of this family are neocarzinostatin, secreted by (Van Roey and Beerman 1989); C-1027 (Xu et al. 1994) and actinoxanthin (Pletnev and Kuzin 1982; Sakata et al. 1993), secreted by (camels and llamas) are an exception, because there are formed only from heavy chains (Hamers-Casterman et al. 1993; Muyldermans et al. 1994). Consequently, in the antigen recognition domain, referred to as VHH, there are only three hypervariable regions, instead of the six antigen-binding loops present in the classical antibodies formed by VH and VL chains. Several VHH/antigen complexes have been crystallized (Desmyter et al. 1996; Spinelli et al. 1996). In the camel VHH/HEWL complex, contact is essentially made via a long Ebselen CDR3 loop of 27 residues (Desmyter et al. 1996). The N-terminal part of this loop (10 residues) penetrates deeply into the active-site cleft of the lysozyme, providing approximately 70% of the antigen contact (Transue et al. 1998). We transferred this sequence to the equivalent site in the corresponding CDR3 Ebselen loop of neocarzinostatin. We studied the stability of the resulting structure and its affinity for hen egg white lysozyme. Results Construction of the NCS-CDR3 NCS has a comparable overall fold with the Ebselen VHH camel immunoglobulin domains. This similarity is clearly exhibited by examination of the topological diagram in Physique 1 ?, which shows the global fold of apo-NCS and the variable VHH domain name of camel antilysozyme Ig. The structure of NCS is usually such that the 99C107 loop located at the surface of the protein could.
