Expression Screening of Human Integral Membrane Proteins Using BacMam

Pravin Mahajan; Katherine Ellis; Shubhashish Mukhopadhyay; Alejandra Fernandez-Cid; Gamma Chi; Henry Man; Katharina L Dürr; Nicola A Burgess-Brown

doi:10.1007/978-1-0716-0892-0_6

Expression Screening of Human Integral Membrane Proteins Using BacMam

Methods Mol Biol. 2021:2199:95-115. doi: 10.1007/978-1-0716-0892-0_6.

Authors

Pravin Mahajan¹, Katherine Ellis², Shubhashish Mukhopadhyay³, Alejandra Fernandez-Cid³, Gamma Chi³, Henry Man³, Katharina L Dürr³, Nicola A Burgess-Brown⁴

Affiliations

¹ Astex Pharmaceuticals, Cambridge, UK.
² Jenner Institute, University of Oxford, Oxford, UK.
³ Structural Genomics Consortium, University of Oxford, Oxford, UK.
⁴ Structural Genomics Consortium, University of Oxford, Oxford, UK. [email protected].

PMID: 33125646
DOI: 10.1007/978-1-0716-0892-0_6

Abstract

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.

Keywords: BacMam; Baculovirus; FSEC; Gel filtration; IMAC; Insect cells; Mammalian cells; Protein purification; SEC chromatography; Transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Expression*
Genetic Vectors / genetics*
HEK293 Cells
Humans
Membrane Proteins* / biosynthesis
Membrane Proteins* / genetics
Recombinant Proteins / biosynthesis
Recombinant Proteins / genetics

Substances

Membrane Proteins
Recombinant Proteins

Grants and funding

WT_/Wellcome Trust/United Kingdom