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

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.

SUBMITTER: Bokhove M

PROVIDER: S-EPMC4771870 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Bokhove Marcel M Sadat Al Hosseini Hamed H Saito Takako T Dioguardi Elisa E Gegenschatz-Schmid Katharina K Nishimura Kaoru K Raj Isha I de Sanctis Daniele D Han Ling L Jovine Luca L

Journal of structural biology 20160203 1

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 e ...[more]

PMID: 26850170

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