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Related Concept Videos

Conjugated Proteins02:50

Conjugated Proteins

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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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Conjugation01:19

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Conjugation is a form of horizontal gene transfer that primarily occurs in bacteria and some archaea, promoting genetic diversity and adaptation. Bacteria can acquire resistance genes through conjugative plasmids, allowing them to survive antibiotic treatments that would otherwise be lethal. This process involves direct contact between cells through specialized structures such as the sex pilus and is mediated by conjugative plasmids, including the F (fertility) factor.Conjugation requires...
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Bacterial conjugation is a mechanism of horizontal gene transfer that enables the exchange of genetic material between bacterial cells through direct contact. This process is facilitated by a donor cell carrying a conjugative plasmid, which encodes genes necessary for pilus formation, DNA replication, and transfer. The conjugative plasmid plays a central role in initiating and executing the transfer of genetic material.The tra region of the conjugative plasmid encodes proteins responsible for...
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What is Natural Selection?01:32

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Natural selection is an evolutionary process in which individuals with survival-promoting traits reproduce at higher rates. These favorable traits become more common within a population or species. Naturally selected traits initially arise via random genetic mutations. In order for selection to occur, there must be variation within a population, the trait controlling the variation must be heritable, and there must be an evolutionary advantage for variation in the trait.
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Relative Strengths of Conjugate Acid-Base Pairs02:29

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Brønsted-Lowry acid-base chemistry is the transfer of protons; thus, logic suggests a relation between the relative strengths of conjugate acid-base pairs. The strength of an acid or base is quantified in its ionization constant, Ka or Kb, which represents the extent of the acid or base ionization reaction. For the conjugate acid-base pair HA / A−, the ionization equilibrium equations and ionization constant expressions are
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Related Experiment Video

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IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II
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Site-selective protein conjugation at histidine.

Karolina Peciak1,2, Emmanuelle Laurine2, Rita Tommasi2

  • 1UCL School of Pharmacy , University College London , 29-39 Brunswick Square , London , WC1N 1AX , UK .

Chemical Science
|February 28, 2019
PubMed
Summary

Site-selective PEGylation of interferon alpha2-a (IFN) was achieved using a histidine tag (His₂-tag). Strategic placement of this tag maximized retained biological activity, yielding an unprecedented 74% for a PEGylated IFN conjugate.

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Area of Science:

  • Bioconjugation Chemistry
  • Protein Engineering
  • Recombinant Protein Expression

Background:

  • Site-selective protein conjugation requires engineered sites and specific chemistries.
  • Polyethylene glycol (PEG) modification of proteins like interferon alpha2-a (IFN) can improve therapeutic properties but often reduces bioactivity.
  • Achieving site-specific PEGylation while preserving protein function is a significant challenge.

Purpose of the Study:

  • To develop a site-selective conjugation strategy for interferon alpha2-a (IFN) using a histidine tag.
  • To investigate the impact of conjugation site location on the biological activity of PEGylated IFN.
  • To optimize the engineering of histidine tags for efficient and functional PEGylation.

Main Methods:

  • Engineered three N-terminal interferon alpha2-a (IFN) variants with truncated histidine tags (His₂-tag and His₃-tag).
  • Utilized site-directed mutagenesis to express IFN variants in E. coli.
  • Performed PEGylation using a bis-alkylation reagent (PEG₁₀kDa-mono-sulfone) and purified PEG-IFN variants via RP-HPLC.

Main Results:

  • Achieved PEGylation conversions ranging from 28-39% with high purity (>97%) and yields (21-33%) after single-step purification.
  • Demonstrated that the site of PEG conjugation significantly impacts retained biological activity.
  • The PEG₁₀-106(HGHG)-IFN variant, with the His₂-tag on a flexible loop distant from the receptor binding site, retained the highest bioactivity (74%).

Conclusions:

  • The His₂-tag is a feasible approach for creating site-selective targets for bis-alkylating PEGylation.
  • Strategic engineering of conjugation sites using His₂-tags can maximize the retention of biological activity in modified proteins.
  • This methodology offers a promising route for developing improved PEGylated protein therapeutics with enhanced efficacy.