Single and dual functionalization of proteins using site-specific nucleophilic carbon ligations.

We here found that while Meldrum's acid as the reactive warhead allows for the attachment of a single chemical modification on aldehyde-containing proteins, pyrazolone derivatives in combination with a phosphine nucleophile enable protein dual site-specific conjugation with the same or distinct moieties. These reactions are efficient and convergent under biocompatible conditions and allow access to protein bioconjugates with superior stability, homogeneity and flexibility. Our work expands the repertoire of bioconjugation chemistries and offers opportunities to construct bioconjugates with defined structure that have potential for medical and biomaterial applications.

Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins.

Here, we report a new protocol for enhancing the soluble expression of inclusion body (IB)-prone proteins in E. coli using nanobodies (Nbs) as a molecular-specific chaperone. The specific intracellular binding between the cognate-Nbs and the antigen is successfully achieved and enables the formation of a soluble Nb-antigen complex in E. coli. We further expand this method by adding an epitope tag (EPEA-tag) to the target proteins, and the anti-EPEA Nb was intended to act as the chaperone for in vivo binding with the EPEA tag. Such substitution may develop a "multi-specific" Nb-chaperone that can simultaneously and effectively cope with different IB proteins of interest.

Cytoplasmic Expression of Nanobodies with Formylglycine Generating Enzyme Tag and Conversion to a Bio-Orthogonal Aldehyde Group.

Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.

Nanobody-loaded immunosorbent for highly-specific removal of interleukin-17A from blood.

Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.55 mg/g gel, which is almost consistent with the saturated adsorption capacity determined in dynamic adsorption. The in vitro plasma perfusion test demonstrated a remarkable adsorptive performance of the Nb-coupled sorbent since more than 75% IL-17A could be eliminated under the plasma/sorbent ratio of 1000:1. These results indicated the Nb-loaded immunosorbent can provide a simple and economic platform technology for immunoaffinity depletion of single or even multiple cytokines from plasma.

One-step Preparation of a VHH-based Immunoadsorbent for the Extracorporeal Removal of ß2-microglobulin.

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of ß2-microglobulin (ß2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of ß2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove ß2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with ß2-m. In this study, we modified the anti-ß2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of ß2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed ß2-m from blood, therefore opening a new avenue for efficient hemodialysis.

Freezing-assisted synthesis of covalent C-C linked bivalent and bispecific nanobodies.

Bi-valent/specific antibodies are coming to the forefront of therapeutic and diagnostic applications for extending the functions of conventional antibodies. Nanobodies as building blocks, due to their small sizes, are prone to synthesizing these homo/hetero-dimers. However, the classical C-terminus to N-terminus (C-N) ligation manner for generating the dimer results in the inhibition of the antigen-binding capacity of the bivalent/specific antibodies. In this study, we designed and constructed several C-terminus to C-terminus (C-C) linked bivalent and bispecific nanobodies against the human ß2-microglobulin via freezing, overcoming the biological function-disrupt raised by the C-N ligation. The nanobody modified by the formylglycine generating enzyme was ligated to a hydrazide or aminooxy bi-functionalized linker. During the process, we discovered that freezing significantly improved the efficiency of hydrazone or oxime formation between the linker and nanobodies, which could not take place at room temperature. By freezing from -10 to -20 °C, up to 50% yield of bivalent nanobodies was achieved within 24 h. The C-C linked nanobody-fusions maintained almost all of its binding activity and exhibited an increase by two orders of magnitudes in affinity kinetics, demonstrating the superiority of C-C over the C-N linking approach.

Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation.

Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.