Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices.

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics.

Development of polyol-responsive antibody mimetics for single-step protein purification.

The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase.

Polydnavirus Ank proteins bind NF-κB homodimers and inhibit processing of Relish.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-κB (NF-κB) transcription factors, and inhibitor κB (IκB) proteins that complex with and regulate NF-κBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs).

Model reactions for insect cuticle sclerotization: participation of amino groups in the cross-linking of Manduca sexta cuticle protein MsCP36.

Current theories of sclerotization center on protein cross-linking and dehydration as major factors in the hardening and stability of the insect cuticle. Several studies have reported the identification of catechol-amino acid adducts from sclerotizing cuticle involving histidine, lysine, and tyrosine, though there have been no reports of a catechol linked between two amino acid residues. Previously, we reported an in vitro model system for sclerotization and observed that stable protein oligomers were formed, presumably through cross-links with oxidized catecholamines [Insect Biochem.

Protein tyrosine phosphatase-H2 from a polydnavirus induces apoptosis of insect cells.

The family Polydnaviridae is a large group of immunosuppressive insect viruses that are symbiotically associated with parasitoid wasps. The polydnavirus Microplitis demolitor bracovirus (MdBV) causes several alterations that disable the cellular and humoral immune defences of host insects, including apoptosis of the primary phagocytic population of circulating immune cells (haemocytes), called granulocytes. Here, we show that MdBV infection causes granulocytes in the lepidopteran Spodoptera frugiperda to apoptose.

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti.

Ingestion of vertebrate blood is essential for egg maturation and transmission of disease-causing parasites by female mosquitoes. Prior studies with the yellow fever mosquito, Aedes aegypti, indicated blood feeding stimulates egg production by triggering the release of hormones from medial neurosecretory cells in the mosquito brain. The ability of bovine insulin to stimulate a similar response further suggested this trigger is an endogenous insulin-like peptide (ILP).

Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols.

The quinone-tanning hypothesis for insect cuticle sclerotization proposes that N-acylcatecholamines are oxidized by a phenoloxidase to quinones and quinone methides, which serve as electrophilic cross-linking agents to form covalent cross-links between cuticular proteins. We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-beta-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M.

Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae.

Laccase (EC 1.10.3.