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Preparation of

Yuqin Wang1,2, Pingping Fan1,2, Shanyu Zhang1,2

  • 1State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.

Biophysics Reports
|June 8, 2023
PubMed

Insights

We present a simplified protocol for preparing Mycobacterium smegmatis porin A (MspA) nanopores, crucial for nanopore sequencing and nano-reactive sensing. This method yields high-quality MspA protein for single-molecule sensing applications.

Area of Science:

  • Biophysics
  • Nanotechnology
  • Molecular Biology

Background:

  • Mycobacterium smegmatis porin A (MspA) offers high spatial resolution for sensing due to its narrow constriction.
  • MspA is ideal for nanopore sequencing and nano-reactive sensing but is not commercially available.
  • Existing MspA preparation methods are complex and inaccessible.

Purpose of the Study:

  • To develop a simplified and accessible protocol for MspA preparation.
  • To demonstrate the utility of prepared MspA nanopores for single-molecule sensing.
  • To enable the development of MspA-based nano-reactive sensors.

Main Methods:

  • A straightforward protocol for MspA protein preparation was developed.
  • The protocol focuses on achieving high yields of fully oligomerized MspA protein.
  • Demonstration of MspA nanopore function using immobilized ssDNA oligonucleotide discrimination.

Main Results:

  • The protocol yields approximately 10 mg of fully oligomerized MspA protein per liter of culture.
  • The prepared MspA nanopores successfully discriminated immobilized ssDNA oligonucleotides.
  • The protocol is compatible with MspA mutants, facilitating sensor development.

Conclusions:

  • A simplified MspA preparation protocol is established, overcoming commercial unavailability.
  • The protocol provides a reliable source of MspA for nanopore sequencing and nano-reactive sensing.
  • This work facilitates the creation of diverse MspA-based nano-sensors for various applications.
Keywords:
Affinity purificationMspA nanoporeProkaryotic expressionSingle molecule sensing

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