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Conjugated Proteins02:50

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Simple proteins and protein complexes contain only amino acids. In contrast, many other proteins, called conjugated proteins, covalently bond with non-protein moieties.
Nucleoproteins are protein complexes that contain nucleic acids, categorized as deoxyribonucleoproteins (DNPs) or ribonucleoproteins (RNPs) respectively. The nucleosome is a typical example of a DNP where nuclear DNA is associated with histone proteins. The major antigen for the Covid-19 virus SARS-CoV is an RNP that is critical...
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Phase II biotransformations are detoxification mechanisms that conjugate xenobiotics with endogenous substances, neutralizing their toxicity.
A key example involves the conjugation of cyanide ions, which impair cellular respiration and alter hemoglobin into non-oxygen-carrying cyanmethemoglobin. To neutralize this threat, a sulfur atom from thiosulphate is transferred to the cyanide ion, catalyzed by the enzyme rhodanese, resulting in an inactive compound called thiocyanate. The production of...
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Phase II Conjugation Reactions: Overview01:14

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Conjugation, a key component of phase II biotransformation reactions, is a vital process in drug detoxification. It involves transferring endogenous substances like glucuronic acid, sulfate, and glycine to drugs or their metabolites formed in phase I reactions. These conjugation reactions, often catalyzed by specific enzymes, transform potentially harmful metabolites into inactive, water-soluble forms easily excreted in urine or bile. By enhancing polarity and eliminating pharmacological...
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Sulfation and α-amino acid conjugation are two critical biotransformation reactions in drug metabolism. Sulfation, a phase II biotransformation reaction, involves adding a polar sulfate group to a drug, enhancing its water solubility and promoting excretion. This process can either co-occur with or occur independently of glucuronidation. Nonmicrosomal sulfotransferase enzymes catalyze the process. The reaction involves 3'-phosphoadenosine-5'-phosphosulfate or PAPS coenzyme...
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Glutathione, a tripeptide made up of glutamate, cysteine, and glycine, is a critical player in the detoxification of drugs and xenobiotics via a process known as glutathione conjugation or mercapturic acid formation. This phase II biotransformation reaction involves the covalent binding of glutathione to a drug or its metabolite, enhancing the compound's water solubility and enabling its excretion.
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Tuning Degradation to Achieve Specific and Efficient Protein Depletion
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Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

Grace H Pham1, Weijia Ou1, Badry Bursulaya1

  • 1Genomics Institute of the Novartis Research Foundation (GNF), Biotherapeutics & Biotechnology, 10675 John Jay Hopkins Drive, San Diego, CA, 92121, USA.

Chembiochem : a European Journal of Chemical Biology
|February 2, 2018
PubMed
Summary

Fluorophenyl esters enable site-selective labeling of antibodies at specific lysine residues. This method enhances protein modification efficiency, offering a valuable tool for antibody engineering and research.

Keywords:
antibodiesbioconjugationchemical toolsflow chemistrylysine

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Protein Engineering

Background:

  • Activated esters are common reagents for protein labeling, targeting lysine side chains and N-termini.
  • Non-selective reactivity of primary amines limits site-specific protein modification.
  • Current methods often lack the precision for targeting single amino acid residues in complex proteins.

Purpose of the Study:

  • To investigate the site-selective labeling of human kappa antibodies using fluorophenyl esters.
  • To identify factors contributing to preferential labeling at specific lysine residues.
  • To optimize labeling conditions for achieving high site-specificity.

Main Methods:

  • Utilized fluorophenyl esters for labeling human kappa antibodies.
  • Analyzed labeling patterns using techniques to identify the specific lysine residue targeted.
  • Investigated the influence of neighboring residues (His189, Asp151) on labeling kinetics.
  • Explored methods to enhance labeling selectivity, including temperature control, flow chemistry, and mutagenesis.

Main Results:

  • Fluorophenyl esters demonstrated preferential labeling of a single lysine residue (Lys188) in human kappa antibodies.
  • Neighboring residues His189 and Asp151 were found to enhance the labeling rate at Lys188.
  • Labeling enrichment at Lys188 was improved from 50-70% to over 95% using optimized conditions.
  • Fluoronaphthyl esters also showed utility for site-selective lysine labeling.

Conclusions:

  • Activated esters with fluoro-substituted aromatic leaving groups are effective for site-selective lysine labeling in antibodies.
  • This approach offers a generalizable strategy for precise modification of immunoglobulin-type proteins.
  • Optimized conditions and protein engineering can significantly enhance labeling specificity, advancing antibody-based research and therapeutics.