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

J Struct Biol.. 2016-04;

Bokhove M, Sadat Al Hosseini H, Saito T, Dioguardi E, Gegenschatz-Schmid K, Nishimura K, Raj I, de Sanctis D, Han L, Jovine L.

Products/Services Used	Details	Operation
PCR Cloning and Subcloning	... replacement. 2. Methods. 2.1. DNA constructs. A DNA fragment encoding mMBP and a multiple cloning site was synthesized (GenScript) and subcloned into pHLsec (Aricescu et al., 2006), resulting in pHLmMBP construct 1 (Fig. S1A). ...	Get A Quote

摘要

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 crys... More