Site-specific Covalent Conjugation Research Articles

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Analytical development insights spanning viral vectors & gene editing

David McCall, Commissioning Editor of Cell & Gene Therapy Insight speaks to Santoshkumar Khatwani, Director Analytical Development, Sangamo Therapeutics, Inc. Dr Santosh Khatwani graduated from the University of Kentucky in 2010 with PhD in Chemistry under the guidance of Prof. Sylvia Daunert. The research work focused on the use of generically engineered recombinant protein and enzymes for bio sensing and biomaterial applications. Furthermore, he obtained postdoctoral training at the University of Minnesota (until 2012) under the supervision of Prof T Andrew Taton and Prof Mark D Distefano with focus on the use of site-specific covalent conjugation for synthesis of protein–DNA conjugates and analytical assays for the generating novel engineered enzymes with improved functional attributes. Dr Khatwani then joined BioVision Inc. in 2012 and served under different capacities until 2017 where he oversaw the manufacture, testing and release of several recombinant protein, enzyme and assays for various metabolically important enzymes. Furthermore, Dr. Khatwani joined the field of cell and gene therapy at multiple companies including Thermofisher Viral Vector Services (formerly Brammer Bio, 2017–2018), Asklepios Biopharmaceuticals (2019) and Sangamo Therapeutics (2018–2019, 2019-present) at various leadership roles where he has led the development of various analytical assays including physicochemical, biophysical and biological assays to support the manufacture and release of viral vectors in cell and gene therapy. Currently Dr. Khatwani is serving as Director of Analytical Development at Sangamo Therapeutics with strong focus on developing analytical solutions and CMC in support of product development at early and late phase of the clinical development.

Non-clinical immunological comparison of a Next-Generation 24-valent pneumococcal conjugate vaccine (VAX-24) using site-specific carrier protein conjugation to the current standard of care (PCV13 and PPV23)

Despite widespread utilization of pneumococcal conjugate vaccines (PCVs) and the resultant disease reduction, the development of PCVs containing additional serotypes remains a public health priority due to serotype replacement and the resultant shift to non-vaccine containing serotypes. However, incorporating additional serotypes to existing PCVs using conventional technologies has proven problematic. Immune responses to individual serotypes have consistently decreased as more polysaccharide-conjugates are added due to carrier suppression. Using our proprietary cell-free protein synthesis (CFPS) platform, we have successfully produced eCRM® based on the CRM197 sequence for use as an enhanced carrier protein to develop a 24-valent PCV. The eCRM carrier protein contains multiple non-native amino acids (nnAAs) located outside of the primary T-cell epitope regions, thereby enabling site-specific covalent conjugation of the pneumococcal polysaccharides to the nnAAs to consistently expose the critical T-cell epitopes. eCRM also serves to reduce structural heterogeneity associated with classic reductive-amination conjugation while promoting formation of the conjugate matrix structures, the hallmark of PCVs. This process serves to increase the overall polysaccharide:protein ratio, enabling the inclusion of more serotypes while minimizing carrier-mediated immunological interference.The aim of this non-clinical study was to construct a 24-valent PCV and evaluate its immunogenicity. Using the XPressCF® CFPS platform, the eCRM carrier protein was separately conjugated through nnAAs to each of the 24 pneumococcal polysaccharides through click chemistry and mixed with aluminum phosphate to produce VAX-24, Vaxcyte’s proprietary PCV preclinical candidate. VAX-24, Prevnar13® and Pneumovax®23 were administered to New Zealand White rabbits to compare the resulting opsonophagocytic activity (OPA) and anti-capsular IgG antibodies. VAX-24 showed conjugate-like immune responses to all 24 serotypes based on comparable OPA and IgG responses to Prevnar13 and higher responses than Pneumovax 23. This study demonstrates the utility of site-specific conjugation technology in a preclinical setting and the potential for a PCV with improved serotype coverage.

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Biocatalysis is an attractive route for the synthesis of complex organic molecules, such as pharmaceuticals, due to the properties of enzymes (high specificity and high catalytic rate). Ideally, we would be able to use enzymes in continuous-flow reactors to benefit from the advantages of continuous-flow chemistry (flexibility, control, product stream purity, low capital cost and improved yields for some reactions). However, continuous-flow applications for biocatalysis face substantial technical obstacles, particularly for enzymes that require cofactors. In the work presented here we tackle two of these obstacles: the provision of cofactor and cofactor recycling in flow, and enzyme immobilization without loss of activity. This is achieved through the production of modular biocatalysts that retain and recycle their cofactors, and that allow orthogonal, site-specific covalent conjugation to a surface. This generalizable engineering approach allowed us to build a complex, multistep flow reactor that outperforms previously published systems for cofactor-dependent continuous-flow biocatalysis. Tackling the loss of expensive cofactors is a key challenge in continuous-flow biocatalysis. This work reports immobilized enzymes with a tethered cofactor that is channelled between a recycling and a catalysis module facilitating total turnover numbers of NAD+ and ATP exceeding 10,000 in biocatalytic flow systems.

Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1β chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1β CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

Abstract 78: Highly efficient antibody drug conjugation with a novel photoactivation strategy

Abstract Meditope SnAP (site-specific novel antibody platform) technology is a novel way to target molecules to antibodies in a site specific, non-destructive fashion. A “meditope” is a proprietary peptide that can bind a meditope-enabled antibody in a unique site and in such a way that antigen binding is not compromised. This specific peptide antibody interaction can be readily employed for site-specific covalent conjugation of cytotoxic payloads to any meditope-enabled antibody. To facilitate covalent conjugation, the meditope peptide has been rationally designed with a photoactivatable side chain and the generation of the antibody-drug conjugate (ADC) occurs in a highly efficient manner following the specific binding of the meditope to the enabled antibody. To test the hypothesis that photoactivation would be an efficacious and homogenous way to make an ADC, meditope-enabled ADCs were designed and tested in vitro and in vivo. Toxin conjugation was tested, as was efficiency of covalent interaction following short-term exposure of the antibody peptide conjugates to a nondestructive wavelength of 340 nM. Photoactivation proved to be a highly efficient and scalable way to make an ADC. Payload stability was confirmed in vivo with multiple toxins and in vivo potency of two unique ADCs were tested in an EGFR positive gastric cancer xenograft as well as a HER2 positive ovarian cancer xenograft. The meditope-enabled photogenerated anti-HER2 ADC was benchmarked against conventionally conjugated T-DM1 and showed equivalent efficacy, but with the site-directed conjugated meditope ADC using less toxin. Meditope-directed photoactivated-controlled conjugation can be readily extended to any meditope-enabled antibody, resulting in a novel way to develop ADCs. This methodology, with its high efficiency, speed and homogeneity has evident advantages for the development of antibody-drug conjugates. Citation Format: Elisabeth Gardiner, Karin Forster, Robert McKenzie. Highly efficient antibody drug conjugation with a novel photoactivation strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2017-78

Improved Performance of Recombinant Protein A Immobilized on Agarose Beads by Site-Specific Conjugation.

Protein A affinity adsorbent with high antibody-binding capacity plays a prominent part in the purification of biopharmaceuticals to decrease the manufacturing costs. We describe a site-specific covalent conjugation strategy for protein A to immobilize on agarose beads. Recombinant protein A, which has one cysteine introduced at the C terminus through genetic engineering technology, was immobilized site-specifically on maleimide-functionalized agarose beads by the thiol–maleimide reaction. As a comparison, the recombinant protein A was randomly immobilized on the aldehyde-functionalized agarose beads via free amino groups on the protein surface. The site-specific conjugation of recombinant protein A on the agarose beads was validated through the assay of free SH groups on the adsorbents using the Ellman’s reagent. Adsorbents containing various amounts of protein A were used to adsorb antibody from human plasma. Analysis of immunoturbidimetry showed that the adsorbed fractions contained the 90.1% IgG, 4.2% IgA, and 5.7% IgM. The maximal antibodies-binding capacities with static adsorption and dynamic adsorption were approximately 64 and 50 mg, respectively, per swollen gram for site-specifically conjugated adsorbent and 31 and 26 mg for randomly conjugated adsorbent. Remarkably, the high antibody-binding capacity for site-specifically conjugated adsorbent outperformed the existing commercial protein A Sepharose (approximately 30 mg/g). The orientation of a protein is crucial for its activity after immobilization, and these results demonstrate that the site-specifically conjugated protein molecule is in a functionally active form to interact with the antibody with weak steric hindrance. The proposed approach may be an attractive strategy to synthesize affinity adsorbents with high-binding capacity.

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.

BackgroundBacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances.In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter.ResultsThermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1–20 or LacZ1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression.The two most performing fusion protein systems namely short thermoinducible LacZ1–8Met-MTGase from NP668/1 and long constitutive LacZ1–8PNP1–20Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ1–8PNP1–20Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain.ConclusionsCombining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ1–8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.

Conjugation of a reactive thiol at the nucleotide binding site for site-specific antibody functionalization.

Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.

Protein Oriented Ligation on Nanoparticles Exploiting O6‐Alkylguanine‐DNA Transferase (SNAP) Genetically Encoded Fusion

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the site-specific covalent conjugation of targeting peptides to nanoparticles. An scFv mutant selective for HER2 tumor antigen is chosen as the targeting ligand. SNAP-scFv is immobilized on magnetofluorescent nanoparticles and its targeting efficiency against HER2-positive cells is assessed by flow cytometry and immunofluorescence.

Multi-enzyme systems: bringing enzymes together in vitro

In living cells enzymes catalyze a wide variety of metabolic processes, which involve multiple reaction steps. Efficient transfer of the intermediates from one catalytic site to the other is achieved by the formation of macromolecular enzyme complexes. This phenomenon, called metabolic channeling, has inspired researchers to bring biocatalysts together in an artificial way as well. This review describes the various in vitro strategies which have been exploited so far. A distinction is made based on the degree of control over the assembly process. Non-specific, covalent co-immobilization as well as non-covalent encapsulation, scaffold-mediated co-localization and site-specific covalent conjugation strategies are discussed.