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

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

Author information

1
Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden.
2
ESRF - The European Synchrotron, Grenoble 38000, France.
3
Karolinska Institutet, Department of Biosciences and Nutrition & Center for Innovative Medicine, Huddinge, Sweden. Electronic address: luca.jovine@ki.se.

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

PMID:
26850170
PMCID:
PMC4771870
DOI:
10.1016/j.jsb.2016.01.016
[Indexed for MEDLINE]
Free PMC Article

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