title: Thermostable single domain antibody-maltose binding protein fusion for Bacillus anthracis spore protein BclA detection.
abstract: 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) ∼ 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.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Walper
authors.affiliation: Clark Atlanta University, Atlanta, GA 30314, USA.
authors.forename: Shawna R
authors.initials: SR
authors.lastname: Battle
authors.affiliation: NOVA Research, Alexandria, VA 22308, USA.
authors.forename: P
authors.initials: P
authors.lastname: Audrey Brozozog Lee
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Kendrick B
authors.initials: KB
authors.lastname: Turner
authors.affiliation: Biosciences Division, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Grounds, Edgewood, MD 21010, USA.
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Buckley
authors.affiliation: Biosciences Division, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Grounds, Edgewood, MD 21010, USA.
authors.forename: Alena M
authors.initials: AM
authors.lastname: Calm
authors.affiliation: Biosciences Division, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Grounds, Edgewood, MD 21010, USA.
authors.forename: Heather S
authors.initials: HS
authors.lastname: Welsh
authors.affiliation: Excet, Springfield, VA 22151, USA.
authors.forename: Candice R
authors.initials: CR
authors.lastname: Warner
authors.affiliation: Excet, Springfield, VA 22151, USA.
authors.forename: Melody A
authors.initials: MA
authors.lastname: Zacharko
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA. Electronic address: george.anderson@nrl.navy.mil.
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics*; D003593:Cytoplasm / Q000235:genetics; D007118:Immunoassay / Q000379:methods*; D058619:Maltose-Binding Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000032:analysis*; D008863:Microspheres; D055550:Protein Stability; D020027:Pyrococcus furiosus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013171:Spores, Bacterial; D013696:Temperature; D044366:Transition Temperature
chemical_list: D019843:Archaeal Proteins; C470593:BclA protein, Bacillus anthracis; D058619:Maltose-Binding Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies
keywords: Immunoassay; Maltose binding protein; Single domain antibody; Thermal stability