Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

J Struct Biol. 2016 Apr;194(1):1-7. doi: 10.1016/j.jsb.2016.01.016. Epub 2016 Feb 3.

Abstract

We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.

Keywords: Crystallization; Glycoproteins; Maltose-binding protein fusion; Mammalian cell expression; Molecular replacement; X-ray crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Crystallography, X-Ray
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Maltose-Binding Proteins / chemistry*
  • Maltose-Binding Proteins / genetics
  • Maltose-Binding Proteins / metabolism
  • Models, Molecular
  • Mutation
  • Protein Conformation*
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sf9 Cells

Substances

  • Bacterial Proteins
  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins