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1.
Bioconjug Chem ; 31(5): 1449-1462, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32302483

RESUMO

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.


Assuntos
Aminas/química , Imidas/química , Proteínas/química , Concentração de Íons de Hidrogênio , Cinética , Lisina/química , Solventes/química , Compostos de Sulfidrila/química , Termodinâmica
2.
Angew Chem Int Ed Engl ; 56(9): 2296-2301, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28133915

RESUMO

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.


Assuntos
Cromatografia de Afinidade/métodos , Proteínas/isolamento & purificação , Biocatálise , Cromatografia de Afinidade/economia , Cromatografia de Afinidade/instrumentação , Desenho de Equipamento , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Isomerases/química , Isomerases/isolamento & purificação , Proteínas Luminescentes/química , Proteínas Luminescentes/isolamento & purificação , Metais/química , Modelos Moleculares , Proteínas/química , Fatores de Tempo , Nicotiana/enzimologia , Proteína Vermelha Fluorescente
3.
Cell Chem Biol ; 29(2): 177-190, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-34921772

RESUMO

Proteases cut with enviable precision and regulate diverse molecular events in biology. Such qualities drive a seemingly inexhaustible appetite for proteases with new activities and capabilities. Comprising 25% of the total industrial enzyme market, proteases appear in consumer goods, such as detergents, textile processing, and numerous foods; additionally, proteases include 25 US Food and Drug Administration-approved medicines and various research tools. Recent advances in protease engineering strategies address target specificity, catalytic efficiency, and stability. This guide to protease engineering surveys best practices and emerging strategies. We further highlight gaps and flexibilities inherent to each system that suggest opportunities for new technology development along with engineered proteases to solve challenges in proteomics, protein sequencing, and synthetic gene circuits.


Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Peptídeo Hidrolases/genética
4.
Sci Rep ; 12(1): 9956, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35705606

RESUMO

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.


Assuntos
Toxinas Botulínicas Tipo A , Clostridium botulinum , Peptídeo Hidrolases , Engenharia de Proteínas , Toxinas Botulínicas Tipo A/química , Catálise , Domínio Catalítico , Clostridium botulinum/enzimologia , Clostridium botulinum/metabolismo , Engenharia de Proteínas/métodos , Especificidade por Substrato
5.
mSphere ; 6(2)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33910993

RESUMO

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.


Assuntos
Anticorpos Antivirais/sangue , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , COVID-19/patologia , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Epitopos/sangue , Epitopos/imunologia , Humanos , Nucleocapsídeo/imunologia , Fosfoproteínas/imunologia , Prognóstico , Fatores de Risco
6.
bioRxiv ; 2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33083803

RESUMO

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within six days post-symptom onset and sometimes within one day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patients comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 Likelihood Ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.

7.
ACS Appl Mater Interfaces ; 11(5): 4757-4765, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30668098

RESUMO

A polymer-based electrode capable of specific detection of human serum albumin, and its glycated derivatives, is described. The sensor is constructed from a glass microscope slide coated with a synthesized, polythiophene film bearing a protected, iminodiacetic acid motif. The electrode surface is then further elaborated to a functional biosensor through deprotection of the iminodiacetic acid, followed by metal-affinity immobilization of a specific and high-affinity, albumin ligand. Albumin was then quantified in buffer and synthetic urine via electrochemical impedance spectroscopy. Glycated albumin was next bound to a boronic acid-modified, single-cysteine dihydrofolate reductase variant to quantify glycation ratios by square-wave voltammetry. The platform offers high sensitivity, specificity, and reproducibility in an inexpensive arrangement. The detection limits exceed the requirements for intermediate-term glycemic control monitoring in diabetes patients at 5 and 1 nM for albumin and its glycated forms, respectively.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Albumina Sérica Humana/urina , Albumina Sérica/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Desenho de Equipamento , Produtos Finais de Glicação Avançada , Humanos , Modelos Biológicos , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Albumina Sérica Glicada
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