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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Single-Molecule Imaging of Nuclear Transport
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Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification.

Miguel Ramirez1, Najla Valdes, Dongli Guan

  • 1Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA.

Protein Engineering, Design & Selection : PEDS
|December 11, 2012
PubMed
Summary

Researchers engineered a Nostoc punctiforme DnaE intein (NpuDnaE) with a single Asp118Gly mutation. This modification enables rapid, C-terminal specific protein cleavage, facilitating efficient protein purification.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Inteins are protein splicing elements that can be engineered for protein manipulation.
  • Traditional intein applications often involve both N-terminal and C-terminal cleavage, limiting specific applications.
  • Developing inteins with predictable and specific cleavage is crucial for advanced protein purification strategies.

Purpose of the Study:

  • To engineer a DnaE intein variant capable of exclusive C-terminal cleavage.
  • To investigate the mechanism underlying the engineered C-terminal specific cleavage.
  • To develop novel protein purification methods utilizing the engineered intein.

Main Methods:

  • Site-directed mutagenesis of the Nostoc punctiforme DnaE intein (NpuDnaE) based on sequence homology.
  • Molecular modeling to elucidate the structural basis of the engineered cleavage activity.
  • Kinetic analysis of the thio-dependent C-terminal cleavage.
  • Development and application of intein-mediated protein purification strategies using elastin-like polypeptide and chitin-binding protein tags.

Main Results:

  • A single Asp118Gly mutation in NpuDnaE suppressed N-terminal cleavage and enhanced C-terminal cleavage efficiency.
  • Molecular modeling indicated that the Asp118 residue normally restricts C-terminal cleavage.
  • The NpuDnaE Asp118Gly mutant demonstrated rapid (80% completion in 3 hours) and specific C-terminal cleavage.
  • Successful application in both column-free and chromatography-based protein purification yielded up to 84 mg/L of electrophoretically pure target protein.

Conclusions:

  • The engineered NpuDnaE Asp118Gly intein provides a powerful tool for specific C-terminal protein cleavage.
  • This intein variant enables efficient and rapid protein purification using various affinity tags.
  • The findings offer a new strategy for recombinant protein purification and biotechnology applications.