RESUMO
We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb-A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K(D) â¼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP-sdAb gave a yield of approximately 100mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb-A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1mg/ml for 1h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1h at 90 °C. The PfuMBP-sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb-A5 both as a capture reagent and as a detection reagent.
Assuntos
Proteínas Arqueais/genética , Imunoensaio/métodos , Proteínas Ligantes de Maltose/genética , Glicoproteínas de Membrana/análise , Proteínas Recombinantes de Fusão/química , Anticorpos de Domínio Único/química , Temperatura , Citoplasma/genética , Microesferas , Estabilidade Proteica , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Esporos Bacterianos , Temperatura de TransiçãoRESUMO
Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.