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Expression Screening of Human Integral Membrane Proteins Using BacMam.

Pravin Mahajan1, Katherine Ellis2, Shubhashish Mukhopadhyay3

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Summary

The Structural Genomics Consortium developed a BacMam system screening method to produce challenging human integral membrane proteins (IMPs). This platform identifies IMP expression and monodispersity using small HEK293 cell volumes.

Keywords:
BacMamBaculovirusFSECGel filtrationIMACInsect cellsMammalian cellsProtein purificationSEC chromatographyTransduction

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Area of Science:

  • Structural biology
  • Biochemistry
  • Molecular biology

Background:

  • Integral membrane proteins (IMPs) are crucial drug targets but challenging to produce.
  • The Structural Genomics Consortium (SGC) requires efficient methods for IMP production.
  • Previous methods were insufficient for highly challenging IMP targets.

Purpose of the Study:

  • To describe the SGC's established screening and production methods for IMPs using the BacMam system.
  • To present a recently developed platform for identifying IMP expression and monodispersity.
  • To optimize IMP production from small volumes of HEK293 cells.

Main Methods:

  • Utilized the BacMam eukaryotic expression system.
  • Established a screening process in 2016 for IMP production.
  • Developed a platform to assess expression and monodispersity of IMPs.
  • Employed small-scale HEK293 cell cultures (3 mL).

Main Results:

  • Successfully screened and produced challenging human IMPs.
  • The developed platform effectively identifies IMP expression.
  • Monodispersity of IMPs was successfully assessed.
  • The method is efficient for small-scale production.

Conclusions:

  • The BacMam system and developed platform are effective for producing challenging human IMPs.
  • This approach enables efficient screening and characterization of IMPs.
  • The methodology is suitable for high-throughput production of IMPs for structural studies.