Abstract
Crystallization chaperones have been used to facilitate the crystallization of challenging proteins. Even though the maltose-binding protein (MBP) is one of the most commonly used crystallization chaperones, the design of optimal expression constructs for crystallization of MBP fusion proteins remains a challenge. To increase the success rate of MBP-facilitated crystallization, a series of expression vectors have been designed with either a short flexible linker or a set of rigid helical linkers. Seven death domain superfamily members were tested for crystallization with this set of vectors, six of which had never been crystallized before. All of the seven targets were crystallized, and their structures were determined using at least one of the vectors. Our successful crystallization of all of the targets demonstrates the validity of our approach and expands the arsenal of the crystallization chaperone toolkit, which may be applicable to crystallization of other difficult protein targets, as well as to other crystallization chaperones.
Highlights
Recombinant expression of fusion proteins containing a target protein and an unrelated expression tags is a common strategy to obtain samples for medical and research applications[1]
There are over 100 deposited maltose-binding protein (MBP) fusion protein structures in the PDB by the middle of 2016, making MBP one of the most utilized crystallization chaperone
There has been no report on the rational design of the linker sequences or systematic crystallization screening of MBP fusion proteins, which may have hindered the wider application of this approach in structural biology
Summary
Recombinant expression of fusion proteins containing a target protein and an unrelated expression tags is a common strategy to obtain samples for medical and research applications[1]. We designed a set of seven expression vectors harboring different helical linker sequences, and selected seven death domains as targets, six of which have never been crystallized before.
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