Simplifying protein expression with ligation-free, traceless and tag-switching plasmids

Synthetic biology and genome-scale protein work both require rapid and efficient cloning, expression and purification. Tools for co-expression of multiple proteins and production of fusion proteins with purification and solubility tags are often desirable. Here we present a survey of plasmid vectors that provide for some of these features with a focus on tools for rapid cloning and traceless tagging – a setup that facilitates removal of fusion tags post-purification leaving behind no ‘scar’ on the final construct. Key features are reviewed, including plasmid replication origins and resistance markers, transcriptional promoters, cloning methods, and fusion tags and their removal by proteolysis. We describe a vector system called pHLIC, which assembles features for simple cloning, overexpression, facile purification, and traceless cleavage, as well as flexibility in modifying the vector to exchange fusion tags.

► Recent plasmid vectors for Escherichia coli protein expression are surveyed.
► Features like replicons, promoters, cloning sites and fusion tags are reviewed.
► Ligation-free cloning methods are compared.
► Tools for traceless cleavage of fusion proteins are highlighted.
► A plasmid that combines recent useful features (pHLIC) is described.