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Related Concept Videos

Gene Evolution - Fast or Slow?02:05

Gene Evolution - Fast or Slow?

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The genomes of eukaryotes are punctuated by long stretches of sequence which do not code for proteins or RNAs. Although some of these regions do contain crucial regulatory sequences, the vast majority of this DNA serves no known function. Typically, these regions of the genome are the ones in which the fastest change, in evolutionary terms, is observed, because there is typically little to no selection pressure acting on these regions to preserve their sequences.
In contrast, regions which code...
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Conservation of Protein Domains Over Different Proteins02:26

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Protein domains are small structurally independent units that are part of a single amino acid chain.  Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
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Leaky Scanning02:28

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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Exon Recombination02:32

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The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes. 
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Mutations in Microorganisms01:18

Mutations in Microorganisms

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Mutations are heritable changes in an organism’s genome involving alterations in the base sequence of DNA or RNA. These changes can influence cellular processes and phenotypic traits, potentially transforming the unaltered wild type into a mutant form. Such changes, termed forward mutations, are pivotal in shaping the genetic diversity of organisms.RNA viruses exhibit the highest mutation rates due to the absence of robust proofreading mechanisms during genome replication. In contrast,...
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Conserved Binding Sites01:49

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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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Related Experiment Video

Updated: Feb 19, 2026

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Directed Evolution of Proteins Based on Mutational Scanning.

Carlos G Acevedo-Rocha1,2,3, Matteo Ferla4, Manfred T Reetz5,6

  • 1Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany. acevedor@gmx.de.

Methods in Molecular Biology (Clifton, N.J.)
|November 1, 2017
PubMed
Summary
This summary is machine-generated.

This study presents an economical, high-throughput protocol for creating scanning mutagenesis libraries. This method aids in engineering protein properties like selectivity and activity, crucial for synthetic organic chemistry and biotechnology.

Keywords:
Cytochrome P450 monooxygenaseDeep mutational scanningDirected evolutionMutability landscapesProtein engineeringSaturation mutagenesisScanning mutagenesisSite-directed mutagenesisStereoselectivitySynthetic biology

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Area of Science:

  • Protein Engineering
  • Synthetic Organic Chemistry
  • Biotechnology

Background:

  • Directed evolution is a powerful protein engineering technique with broad applications.
  • The success of directed evolution is often limited by the throughput of screening systems.
  • Developing smaller, smarter libraries is essential when high-throughput screening is not feasible.

Purpose of the Study:

  • To provide an economical, high-throughput protocol for constructing scanning mutagenesis libraries.
  • To guide directed evolution by understanding sequence-function relationships through mutability landscapes.
  • To engineer activity, regioselectivity, and stereoselectivity in cytochrome P450 enzymes for steroid hydroxylation.

Main Methods:

  • Development of a protocol for economical scanning mutagenesis library construction.
  • Utilizing cytochrome P450 enzymes as a model system.
  • Employing mutability landscape concepts to guide library design.

Main Results:

  • A high-throughput method for generating scanning mutagenesis libraries was established.
  • The protocol enables the engineering of specific protein traits, including selectivity and activity.
  • The method is applicable to challenging reactions like steroid oxidative hydroxylation.

Conclusions:

  • The developed protocol offers an efficient approach for creating targeted protein variant libraries.
  • This method facilitates advancements in protein engineering for both basic research and industrial applications.
  • The protocol is valuable for deep mutational scanning experiments and engineering enzyme function.