Sortase A: A Model for Transpeptidation and Its Biological Applications.

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

The Complete Assessment of Small Molecule and Peptidomimetic Inhibitors of Sortase A Towards Antivirulence Treatment.

This review covers the most recent advances in the development of inhibitors for the bacterial enzyme sortase A (SrtA). Sortase A (SrtA) is a critical virulence factor, present ubiquitously in Gram-positive bacteria of which many are pathogenic. Sortases are key enzymes regulating bacterial adherence to host cells, by anchoring extracellular matrix-binding proteins to the bacterial outer cell wall. By targeting virulence factors, effective treatment can be achieved, without inducing antibiotic resistance to the treatment. This is a potentially more sustainable, long-term approach to treating bacterial infections, including ones that display multiple resistance to current therapeutics. There are many promising approaches available for SrtA inhibition, some of which have the potential to advance into further clinical development, with peptidomimetic and in vivo active small molecules being among the most promising. There are currently no approved drugs on the market targeting SrtA, despite its promise, adding to the relevance of this review article, as it extends to the pharmaceutical industry additionally to academic researchers.

An approach for the intracellular delivery of IgG via enzymatic ligation with a cell-permeable attenuated cationic amphiphilic lytic peptide.

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Halenaquinol Blocks Staphylococcal Protein A Anchoring on Cell Wall Surface by Inhibiting Sortase A in Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1-6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC50 of 13.94 µM (4.66 µg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC50 of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence.

Synthesis, Antimicrobial Activities, and Molecular Modeling Studies of Agents for the Sortase A Enzyme.

Sortase A (SrtA) is an attractive target for developing new anti-infective drugs that aim to interfere with essential virulence mechanisms, such as adhesion to host cells and biofilm formation. Herein, twenty hydroxy, nitro, bromo, fluoro, and methoxy substituted chalcone compounds were synthesized, antimicrobial activities and molecular modeling strategies against the SrtA enzyme were investigated. The most active compounds were found to be T2, T4, and T19 against Streptococcus mutans (S. mutans) with MIC values of 1.93, 3.8, 3.94 µg/mL, and docking scores of -6.46, -6.63, -6.73 kcal/mol, respectively. Also, these three active compounds showed better activity than the chlorohexidine (CHX) (MIC value: 4.88 µg/mL, docking score: -6.29 kcal/mol) in both in vitro and in silico. Structural stability and binding free energy analysis of S.mutans SrtA with active compounds were measured by molecular dynamic (MD) simulations throughout 100 nanoseconds (ns) time. It was observed that the stability of the critical interactions between these compounds and the target enzyme was preserved. To prove further, in vivo biological evaluation studies could be conducted for the most promising precursor compounds T2, T4, and T19, and it might open new avenues to the discovery of more potent SrtA inhibitors.

Affinity Resins for the Isolation of Immunoglobulins G Obtained Using Biocatalytic Technology.

Affinity chromatography resins that are obtained by conjugation of matrices with proteins of bacterial origin, like protein A, are frequently used for the purification of numerous therapeutic monoclonal antibodies. This article presents the development of a biocatalytic method for the production of novel affinity resins with an immobilized mutant form of protein A via sortase A mediated reaction. The conditions for activation of the agarose Seplife 6FF matrix, selection of different types of linkers with free amino groups and conditions for immobilization of recombinant protein A on the surface of the activated matrix were studied. Finally, the basic operational properties, like dynamic binding capacity (DBC), temperature dependance of DBC and stability during the cleaning-in-place process of the affinity resin with the Gly-Gly-EDA-Gly-Gly linker, were assessed using recombinant hyperchimeric monoclonal antibodies. The main characteristics show comparable results with the widely used commercial samples.

Wogonin attenuates the pathogenicity of Streptococcus pneumoniae by double-target inhibition of Pneumolysin and Sortase A.

Streptococcus pneumoniae (S. pneumoniae) is a major causative agent of respiratory disease in patients and can cause respiratory distress and other symptoms in severe cases. Pneumolysin (PLY) is a pore-forming toxin that induces host tissue injury and inflammatory responses. Sortase A (SrtA), a catalytic enzyme that anchors surface-associated virulence factors, is critical for S. pneumoniae virulence. Here, we found that the active ingredient of the Chinese herb Scutellaria baicalensis, wogonin, simultaneously inhibited the haemolytic activity of PLY and SrtA activity. Consequently, wogonin decreased PLY-mediated cell damage and reduced SrtA-mediated biofilm formation by S. pneumoniae. Furthermore, our data indicated that wogonin did not affect PLY expression but directly altered its oligomerization, leading to reduced activity. Furthermore, the analysis of a mouse pneumonia model further revealed that wogonin reduced mortality in mice infected with S. pneumoniae laboratory strain D39 and S. pneumoniae clinical isolate E1, reduced the number of colony-forming units in infected mice and decreased the W/D ratio and levels of the inflammatory factors TNF-α, IL-6 and IL-1ß in the lungs of infected mice. Thus, wogonin reduces S. pneumoniae pathogenicity by inhibiting the dual targets PLY and SrtA, providing a treatment option for S. pneumoniae infection.

Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.

NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.

Echinacoside, a promising sortase A inhibitor, combined with vancomycin against murine models of MRSA-induced pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for a range of severe infections, such as skin infections, bacteremia, and pneumonia. Due to its antibiotic-resistant nature, current research focuses on targeting its virulence factors. Sortase A (SrtA) is a transpeptidase that anchors surface proteins to the bacterial cell wall and is involved in adhesion and invasion to host cells. Through fluorescence resonance energy transfer (FRET), we identified echinacoside (ECH), a natural polyphenol, as a potential SrtA inhibitor with an IC50 of 38.42 µM in vitro. It was demonstrated that ECH inhibited SrtA-mediated S. aureus fibrinogen binding, surface protein A anchoring, and biofilm formation. The fluorescence quenching assay determined the binding mode of ECH to SrtA and calculated the KA-binding constant of 3.09 × 105 L/mol, demonstrating the direct interaction between the two molecules. Molecular dynamics simulations revealed that ECH-SrtA interactions occurred primarily at the binding sites of A92G, A104G, V168A, G192A, and R197A. Importantly, the combination of ECH and vancomycin offered protection against murine models of MRSA-induced pneumonia. Therefore, ECH may serve as a potential antivirulence agent against S. aureus infections, either alone or in combination with vancomycin.

A dual biomarker-targeting probe enables signal-on surface labeling of Staphylococcus aureus.

Imaging or killing of a specific pathogen is of significance for precise therapy. Staphylococcus aureus (S. aureus) is an infectious gram-positive bacteria relying on Sortase A (SrtA) to anchor cell surface protein on peptidoglycan. We herein report signal-on labeling of S. aureus with self-quenched optical probes featuring vancomycin-conjugated SrtA substrate that is flanked by a dabcyl moiety paired with either fluorescein or eosine photosensizer (PS). SrtA-mediated cleavage of the substrate motif releases the dabcyl quencher, leading to covalent labeling of peptidoglycan with fluorescein or PS of restored photophysical property. The dual biomarked-enabled peptidoglycan labeling enables signal-on imaging and effective photodynamic destruction of S. aureus, suggesting a protheranostic approch activatable to SrtA-positive bacteria engaged in myriad diseases.

Oroxylin a glucuronide as a novel class of reversible inhibitors of Sortase a, combats MRSA-induced infections.

AIMS: The main purpose of this study was to study the therapeutical effect of oroxylin A glucuronide (OAG) on methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: By substrate peptide reaction-based fluorescence resonance energy transfer (FRET) screening, we identified that OAG was an efficient inhibitor of Sortase A (SrtA) with an IC50 of 45.61 µg mL-1, and achieved efficacy in the treatment of Staphylococcus aureus (S. aureus) infections. We further demonstrated that OAG inhibited the adhesion of the S. aureus to fibrinogen, the surface protein A anchoring and diminished biofilm formation. Results obtained from fluorescence quenching assay elucidated a direct interaction between OAG and SrtA. Employing molecular dynamics simulations, we proved that OAG binds to the binding sites of R197, G192, E105, and V168 in the SrtA. Notably, OAG exhibited a robust therapeutic effect in a MRSA-induced pneumonia model. CONCLUSIONS: We identified that OAG as a novel class of reversible inhibitors of SrtA, combats MRSA-induced Infections.

Investigation of the inhibitory effect and mechanism of epigallocatechin-3-gallate against Streptococcus suis sortase A.

AIMS: Streptococcus suis seriously harms people and animals, and importantly, causes great economic losses in the pig industry. Similar to most Gram-positive pathogenic bacteria, sortase A (SrtA) of S. suis can mediate the anchoring of a variety of virulence factors that contain specific sorting sequences to the surface of the bacterial cell wall envelope and participate in pathogenicity. The purpose of this study is to clarify the molecular mechanism of epigallocatechin-3-gallate (EGCG) inhibiting S. suis SrtA and provide more evidence for the development of novel anti-S. suis infections drugs. METHODS AND RESULTS: Through the SrtA substrate cleavage experiment, we found that the main component of green tea, EGCG, can effectively inhibit the enzyme activity of S. suis SrtA. Further, molecular docking and molecular dynamics simulation were used to clarify the molecular mechanism of its inhibitory effect, demonstrating that EGCG mainly interacts with amino acids at 113 and 115 to exert its inhibitory function. It was previously found that EGCG can inhibit the growth of S. suis and reduce the activity of suilysin and inhibit its expression. Our research reveals a new function of EGCG in S. suis infection. CONCLUSIONS: Our research proves that EGCG can effectively inhibit the transpeptidase activity of SrtA. We also clarify the accompanying molecular mechanism, providing more sufficient evidence for the use of EGCG as a potential lead compound against S. suis infection.

Development of new spiro[1,3]dithiine-4,11'-indeno[1,2-b]quinoxaline derivatives as S. aureus Sortase A inhibitors and radiosterilization with molecular modeling simulation.

Multi-drug resistant microbes have become a severe threat to human health and arise a worldwide concern. A total of fifteen spiro-1,3-dithiinoindenoquinoxaline derivatives 2-7 were synthesized and evaluated for their biological activities against five standard and MDRB pathogens. The MIC and MBC/MFC for the most active derivatives were determined in vitro via broth microdilution assay. These derivatives showed significant activity against the tested strains with microbicidal behavior, with compound 4b as the most active compound (MIC range between 0.06 and 0.25 µg/mL for bacteria strains and MIC = 0.25 µg/mL for C. albicans). The most active spiro-1,3-dithiinoindenoquinoxaline derivatives were able to inhibit the activity of SrtA with IC50 values ranging from 22.15 ± 0.4 µM to 37.12 ± 1.4 µM. In addition, the active spiro-1,3-dithiinoindenoquinoxaline attenuated the in vitro virulence-related phenotype of SrtA by weakening the adherence of S. aureus to fibrinogen and reducing the biofilm formation. Surprisingly, compound 4b revealed potent SrtA inhibitory activity with IC50 = 22.15 µM, inhibiting the adhesion of S. aureus with 39.22 ± 0.15 % compared with untreated 9.43 ± 1.52 %, and showed a reduction in the biofilm biomass of S. aureus with 32.27 ± 0.52 %. We further investigated the effect of gamma radiation as a sterilization method on the microbial load and found that a dose of 5 kGy was sufficient to eradicate the microbial load. The quantum chemical studies exhibited that the tested derivatives have a small energy band gap (ΔE = -2.95 to -3.61 eV) and therefore exert potent bioactivity by interacting with receptors more stabilizing.

New Cyclopiane Diterpenes and Polyketide Derivatives from Marine Sediment-Derived Fungus Penicillium antarcticum KMM 4670 and Their Biological Activities.

Two new cyclopiane diterpenes and a new cladosporin precursor, together with four known related compounds, were isolated from the marine sediment-derived fungus Penicillium antarcticum KMM 4670, which was re-identified based on phylogenetic inference from ITS, BenA, CaM, and RPB2 gene regions. The absolute stereostructures of the isolated cyclopianes were determined using modified Mosher's method and quantum chemical calculations of the ECD spectra. The isolation from the natural source of two biosynthetic precursors of cladosporin from a natural source has been reported for the first time. The antimicrobial activities of the isolated compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans as well as the inhibition of staphylococcal sortase A activity were investigated. Moreover, the cytotoxicity of the compounds to mammalian cardiomyocytes H9c2 was studied. As a result, new cyclopiane diterpene 13-epi-conidiogenone F was found to be a sortase A inhibitor and a promising anti-staphylococcal agent.

Anthraquinone Derivatives and Other Aromatic Compounds from Marine Fungus Asteromyces cruciatus KMM 4696 and Their Effects against Staphylococcus aureus.

New anthraquinone derivatives acruciquinones A-C (1-3), together with ten known metabolites, were isolated from the obligate marine fungus Asteromyces cruciatus KMM 4696. Acruciquinone C is the first member of anthraquinone derivatives with a 6/6/5 backbone. The structures of isolated compounds were established based on NMR and MS data. The absolute stereoconfigurations of new acruciquinones A-C were determined using ECD and quantum chemical calculations (TDDFT approach). A plausible biosynthetic pathway of the novel acruciquinone C was proposed. Compounds 1-4 and 6-13 showed a significant antimicrobial effects against Staphylococcus aureus growth, and acruciquinone A (1), dendryol B (4), coniothyrinone B (7), and ω-hydroxypachybasin (9) reduced the activity of a key staphylococcal enzyme, sortase A. Moreover, the compounds, excluding 4, inhibited urease activity. We studied the effects of anthraquinones 1, 4, 7, and 9 and coniothyrinone D (6) in an in vitro model of skin infection when HaCaT keratinocytes were cocultivated with S. aureus. Anthraquinones significantly reduce the negative impact of S. aureus on the viability, migration, and proliferation of infected HaCaT keratinocytes, and acruciquinone A (1) revealed the most pronounced effect.

Dual-Warhead Conjugate Based on Fibroblast Growth Factor 2 Dimer Loaded with α-Amanitin and Monomethyl Auristatin E Exhibits Superior Cytotoxicity towards Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1.

Targeting fibroblast growth factor receptor 1 (FGFR1) is a promising therapeutic strategy for various cancers associated with alterations in the FGFR1 gene. In this study, we developed a highly cytotoxic bioconjugate based on fibroblast growth factor 2 (FGF2), which is a natural ligand of this receptor, and two potent cytotoxic drugs-α-amanitin and monomethyl auristatin E-with completely independent mechanistic modes of action. Utilizing recombinant DNA technology, we produced an FGF2 N- to C-end dimer that exhibited superior internalization capacity in FGFR1-positive cells. The drugs were site-specifically attached to the targeting protein using SnoopLigase- and evolved sortase A-mediated ligations. The resulting dimeric dual-warhead conjugate selectively binds to the FGFR1 and utilizes receptor-mediated endocytosis to enter the cells. Moreover, our results demonstrate that the developed conjugate exhibits about 10-fold higher cytotoxic potency against FGFR1-positive cell lines than an equimolar mixture of single-warhead conjugates. The diversified mode of action of the dual-warhead conjugate may help to overcome the potential acquired resistance of FGFR1-overproducing cancer cells to single cytotoxic drugs.

Sortase A Inhibitor Protein Nanoparticle Formulations Demonstrate Antibacterial Synergy When Combined with Antimicrobial Peptides.

Sortase A (SrtA) is an enzyme which attaches proteins, including virulence factors, to bacterial cell walls. It is a potential target for developing anti-virulence agents against pathogenic and antimicrobial resistant bacteria. This study aimed to engineer 𝛽-lactoglobulin protein nanoparticles (PNPs) for encapsulating safe and inexpensive natural SrtA inhibitors (SrtAIs; trans-chalcone (TC), curcumin (CUR), quercetin (QC), and berberine (BR)) to improve their poor aqueous dispersibility, to screen for synergy with antimicrobial peptides (AMPs), and to reduce the cost, dose, and toxicity of AMPs. Minimum inhibitory concentration (MIC), checkerboard synergy, and cell viability assays were performed for SrtAI PNPs against Gram-positive (methicillin-sensitive and -resistant S. aureus) and Gram-negative (E. coli, P. aeruginosa) bacteria alone and combined with leading AMPs (pexiganan, indolicidin, and a mastoparan derivative). Each SrtAI PNP inhibited Gram-positive (MIC: 62.5-125 µg/mL) and Gram-negative (MIC: 31.3-500 µg/mL) bacterial growth. TC PNPs with pexiganan demonstrated synergy against each bacteria, while BR PNPs with pexiganan or indolicidin provided synergy towards S. aureus. Each SrtAI PNP inhibited SrtA (IC50: 25.0-81.8 µg/mL), and did not affect HEK-293 cell viability at their MIC or optimal synergistic concentrations with AMPs. Overall, this study provides a safe nanoplatform for enhancing antimicrobial synergy to develop treatments for superbug infections.

Punicalagin, an Inhibitor of Sortase A, Is a Promising Therapeutic Drug to Combat Methicillin-Resistant Staphylococcus aureus Infections.

Antimicrobial resistance (AMR) poses a major threat to human health globally. Staphylococcus aureus is recognized as a cause of disease worldwide, especially methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). The enzyme sortase A (SrtA), present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability, and SrtA-deficient S. aureus strains do not affect the growth of bacteria. Here, we found that punicalagin, a natural compound, was able to inhibit SrtA activity with a very low half maximal inhibitory concentration (IC50) value of 4.23 µg/mL, and punicalagin is a reversible inhibitor of SrtA. Moreover, punicalagin has no distinct cytotoxicity toward A549, HEK293T, or HepG2 cells at a much higher concentration than the IC50 detected by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays. In addition, punicalagin visibly attenuated the virulence-related phenotype of SrtA in vitro by decreasing adhesion of S. aureus to fibrinogen, reducing the ability of protein A (SpA) displayed on the surface of the bacteria and biofilm formation. Fluorescence quenching elucidated the interaction between punicalagin and SrtA. Molecular docking further implied that the inhibitory activity lay in the bond between punicalagin and SrtA residues LYS190, TYR187, ALA104, and GLU106. In In vivo studies, we surprisingly found that punicalagin had a more effective curative effect combined with cefotaxime when mice were infected with pneumonia caused by MRSA. Essentially, punicalagin, a therapeutic compound targeting SrtA, demonstrates great potential for combating MRSA infections.

Production of pentaglycine-fused proteins using Escherichia coli expression system without in vitro peptidase treatment.

Conjugation of functional molecules to peptides is necessary for protein analysis and applications. Transpeptidase sortase A catalyzes the ligation reaction between the amino acid sequence LPXTG and polyglycine and allows for peptide sequence-specific molecular modifications. In this study, the preparation of pentaglycine-fused green fluorescent protein (G5-GFP) via methionine truncation mediated by Escherichia coli endogenous methionyl aminopeptidase was investigated. Some expression vectors of GFP presenting MetGly5 at the N-terminal were constructed, and N-terminal sequence analyses of the protein expressed in E. coli were performed. When the first codon of the GFP-encoding sequence was AUG, a mixture of GFP without pentaglycine and G5-GFP was obtained. In contrast, when the first codon AUG was replaced with a codon encoding alanine, G5-GFP was obtained uniformly. These results showed that the location of AUG in the expression vector had a significant influence on the preparation of polyglycine-fused proteins. The obtained findings are useful for the preparation of polyglycine-fused substrates using E. coli.