Enzyme-mediated Ligation Research Articles

When Supramolecular Chemistry Meets Chemical Biology: New Strategies to Target Proteins through Host-Guest Interactions.

Supramolecular chemistry for targeting proteins is of great interest for the development of novel approaches to recognize, isolate and control proteins. Taking advantage of chemical biology approaches, such as genetic-code expansion and enzyme-mediated ligation, guest recognition elements can be built into proteins of interest, allowing supramolecular control of protein function and regulation. In this viewpoint article, we will discuss the methods, applications, limitations, and future perspectives of supramolecular chemistry for targeting proteins in a site-specific manner.

Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine.

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A Streptococcus (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced TH1/TH2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.

A Self-Adjuvanting Vaccine Platform: Optimization of Site-Specific Sortase A Mediated Conjugation of Toll-Like Receptor 2 Ligands onto the Carboxyl or Amino terminus of Recombinant Protein Antigens.

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a β-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the β-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50 <1nM).

Creating Rechargeable Antithrombotic Surfaces for Implantation via Enzyme-Mediated Ligation: A Novel System for Keeping Medical Devices Functional

Creating Rechargeable Antithrombotic Surfaces for Implantation via Enzyme-Mediated Ligation: A Novel System for Keeping Medical Devices Functional

A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics.

A versatile method is described to engineer precisely defined protein/peptide-polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin-like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme-mediated ligation; and 3) attachment of a polymer by a click reaction with near-quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water-soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide-polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications.

Delivery of Non-Native Cargo into Mammalian Cells Using Anthrax Lethal Toxin.

The intracellular delivery of peptide and protein therapeutics is a major challenge due to the plasma membrane, which acts as a barrier between the extracellular environment and the intracellular milieu. Over the past two decades, a nontoxic PA/LFN delivery platform derived from anthrax lethal toxin has been developed for the transport of non-native cargo into the cytosol of cells in order to understand the translocation process through a protective antigen (PA) pore and to probe intracellular biological functions. Enzyme-mediated ligation using sortase A and native chemical ligation are two facile methods used to synthesize these non-native conjugates, inaccessible by recombinant technology. Cargo molecules that translocate efficiently include enzymes from protein toxins, antibody mimic proteins, and peptides of varying lengths and non-natural amino acid compositions. The PA pore has been found to effectively convey over 30 known cargos other than native lethal factor (LF; i.e., non-native) with diverse sequences and functionalities on the LFN transporter protein. All together these studies demonstrated that non-native cargos must adopt an unfolded or extended conformation and contain a suitable charge composition in order to efficiently pass through the PA pore. This review provides insight into design parameters for the efficient delivery of new cargos using PA and LFN.

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.

Multiple chemical ligation under thermal cycle

Enzymatic ligation methods are useful in diagnostic detection of DNA sequence. Here we describe the investigation of nonezymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for detection and identification of RNA and DNA. Specificity of ligation on DNA target is shown to yield discrimination of single point mutation as a drop in two magnitude. Although enzymatic ligation shows very low activity for RNA target, this reaction is found to be very efficient on RNA target. This chemical ligation with RNA target completes 70% within a few seconds, which equal or overcome ligase enzyme-mediated ligation with DNA target. The reaction is also shown to exhibit a significant level of signal amplification under thermal cycle for short time. Further, we found recently that ligation fidelity changed in function of chemical reactivity of probe. This trend was systematically investigated.

Template-directed photoligation of oligodeoxyribonucleotides via 4-thiothymidine.

Non-enzymatic, template-directed ligation of oligonucleotides in aqueous solution has been of great interest because of its potential synthetic and biomedical utility and implications for the origin of life. Though there are many methods for template-directed chemical ligation of oligonucleotides, there are only three reported photochemical methods. In the first report, template-directed photoligation was effected by cyclobutane dimer formation between the 5'- and 3'-terminal thymidines of two oligonucleotides with >290 nm light, which also damages DNA itself. To make the photochemistry of native DNA more selective, we have replaced the thymidine at the 5'-end of one oligonucleotide with 4-thiothymidine (s4T) and show that it photoreacts at 366 nm with a T at the 3'-endof another oligonucleotide in the presence of a complementary template. When a single mismatch is introduced opposite either the s4T or its adjoining T, the ligation efficiency drops by a factor of five or more. We also show that by linking the two ends of the oligonucleotides together, photoligation can be used to form circular DNA molecules and to 'photopadlock' circular DNA templates. Thus, s4T-mediated photo-ligation may have applications to phototriggered antisense-based or antigene-based genetic tools, diagnostic agents and drugs, especially for those situations in which chemical or enzyme-mediated ligation isundesirable or impossible, for example inside a cell.