Scott A. Walper

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

Bacterial Nanobioreactors–Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles

All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

Understanding How Nanoparticle Attachment Enhances Phosphotriesterase Kinetic Efficiency

As a specific example of the enhancement of enzymatic activity that can be induced by nanoparticles, we investigate the hydrolysis of the organophosphate paraoxon by phosphotriesterase (PTE) when the latter is displayed on semiconductor quantum dots (QDs). PTE conjugation to QDs underwent extensive characterization including structural simulations, electrophoretic mobility shift assays, and dynamic light scattering to confirm orientational and ratiometric control over enzyme display which appears to be necessary for enhancement. PTE hydrolytic activity was then examined when attached to ca. 4 and 9 nm diameter QDs in comparison to controls of freely diffusing enzyme alone. The results confirm that the activity of the QD conjugates significantly exceeded that of freely diffusing PTE in both initial rate (∼4-fold) and enzymatic efficiency (∼2-fold). To probe kinetic acceleration, various modified assays including those with increased temperature, presence of a competitive inhibitor, and increased viscosity were undertaken to measure the activation energy and dissociation rates. Cumulatively, the data indicate that the higher activity is due to an acceleration in enzyme-product dissociation that is presumably driven by the markedly different microenvironment of the PTE-QD bioconjugates hydration layer. This report highlights how a specific change in an enzymatic mechanism can be both identified and directly linked to its enhanced activity when displayed on a nanoparticle. Moreover, the generality of the mechanism suggests that it could well be responsible for other examples of nanoparticle-enhanced catalysis.

Comparison of single domain antibody immobilization strategies evaluated by surface plasmon resonance

The use of single domain antibodies (sdAbs) in place of conventional antibodies for both therapeutic and diagnostic applications continues to grow. SdAbs offer a number of advantages when compared to conventional antibodies such as their small size and low structural complexity which allows them to readily be produced as fusions in a variety formats. In this work we compared the utility of various C-terminal fusions and immobilization strategies for two sdAbs; one which recognizes ricin and the other EA1, an S-layer protein, of Bacillus anthracis. Comparisons were made between direct covalent attachment and affinity immobilization using a biotin-streptavidin interaction for the standard sdAb monomers, randomly and site-specifically biotinylated monomers, and fusion constructs of alkaline phosphatase dimers and streptavidin core tetramers. The sdAb binding and regeneration was evaluated by surface plasmon resonance in a multiplexed format. The construct that provided the highest density of active molecules by at least a factor of two was the sdAb-streptavidin core tetramer, followed by the sdAb-alkaline phosphatase and then the site-specifically biotinylated monomer. The poorest performing immobilization methods were the two most common, direct covalent attachment and the randomly biotinylated sdAb attached via NeutrAvidin. These improvements directly correlated to antigen capture in SPR assays. Similarly, the oriented immobilization method also translated to improvements in limit of detection assays using a bead-based system. The sdAb-streptavidin core provided more than a 100-fold improvement in the limit of detection of EA1, from ~200 ng/mL to to 1.6 ng/mL, while improvement for ricin detection was less but still a significant 5-fold decrease, going from 1.6 ng/mL down to 0.32 ng/mL. This demonstrated improvement in limits of detection is an advantage that should be transferable to most assay formats.

Llama-derived single domain antibodies specific for Abrus agglutinin.

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations.

Negative tail fusions can improve ruggedness of single domain antibodies

Single-domain antibodies (sdAbs), the recombinantly expressed binding domains derived from the heavy-chain-only antibodies found in camelids and sharks, are valued for their ability to refold after heat denaturation. However, some sdAbs are prone to aggregation on extended heating at high concentration. Additionally, sdAbs prepared cytoplasmically often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. Genetic fusions of sdAbs with the acid tail of α-synuclein (ATS) resulted in constructs that had enhanced ability to resist aggregation. In addition, almost complete refolding was observed even in the absence of the disulfide bond. These sdAb-ATS fusions expand the utility of sdAbs. They provide sdAbs that are resistant to aggregation, and enable the production of re-foldable sdAbs in the reducing environment of the cytoplasm.

Enzymatic bioconjugation of nanoparticles: developing specificity and control.

Nanoparticles are finding increasing roles in biotechnology for applications as contrast agents, probes, sensors, therapeutics and increasingly new value-added hybrid materials such as molecular logic devices. In most cases these materials must be conjugated to different types of biologicals such as proteins or DNA to accomplish this. However, most traditional methods of bioconjugation result in heterogeneous attachment and loss of activity. Bioorthogonal chemistries and in particular enzymatic labeling chemistries offer new strategies for catalyzing specific biomolecular attachment. We highlight current enzymatic labeling methods available for bioconjugating nanoparticles, some materials they have been used with, and how the resulting bioconjugates were applied. A discussion of the benefits and remaining issues associated with this type of bioconjugation chemistry and a brief perspective on how this field will develop is also provided.

Kinetic enhancement of the diffusion-limited enzyme beta-galactosidase when displayed with quantum dots

The phenomenon of enzyme catalytic enhancement when displayed on a nanoparticle surface has now become well established in the literature and has significant implications across many disciplines that rely on enzymes. It is thus essential to determine which enzymes best utilize this effect. Of particular interest is β-galactosidase, one of the best characterized enzymes which typically operates optimally at diffusion-limited rates. We couple this large tetrameric enzyme to semiconductor quantum dots (QDs) by utilizing a configuration opposite to that used previously with the QDs now displayed on the enzyme. Following physicochemical characterization of the resulting hybrid conjugates, we compared its activity to that of free enzyme. We find that, despite anticipating a seemingly hard rate ceiling, the presence of QDs around the enzyme surface results in a three-fold enhancement of catalytic rate (kcat), suggesting a super-diffusional rate of substrate accessibility. Mechanisms that may account for these results are discussed along with potential applications for utilizing this type of enhanced catalytic configuration.

Bioconjugates of rhizavidin with single domain antibodies as bifunctional immunoreagents

Use of the avidin-biotin binding interaction for immunoassay applications is widespread. One advantageous immunoreagent is the recombinant fusion of an antibody fragment with a biotin binding protein. These genetic fusions alleviate the need to prepare chemical conjugates to achieve molecules that combine target recognition with signal transduction or to facilitate the directional immobilization of the binding element. In order for such a fusion protein to be useful, however, it must be able to be produced in good yield. Unfortunately, recombinant production of avidin or streptavidin as well as bioconjugates derived thereof has been problematic. An alternative biotin binding molecule called rhizavidin has been described, which forms a homodimer instead of a tetramer, but it has not been evaluated in genetic fusions with antibody binding domains. Single domain antibodies, the variable domain derived from camelid heavy chain only antibodies, offer binding domains with high affinity, and solubility that are well expressed in Escherichia coli. In this work, we prepared an anti-ricin single domain antibody - rhizavidin bioconjugate and evaluated it on the basis of its production in E. coli and on its activity in comparison to a streptavidin core bioconjugate and unfused single domain antibody. The single domain antibody-rhizavidin bioconjugate produced much better than its streptavidin core counterparts, yielding an average of 14 mg/L, a 20-fold improvement. When used in assays the rhizavidin conjugate provided the same desirable characteristics as the streptavidin core fusion as both capture and detection reagents. Since rhizavidin and single domain antibodies both display impressive thermal stabilities their fusion provides a route to achieve robust bifunctional immunoreagents.

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

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.