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.
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA. vdurani@chemistry.ohio-state.edu
Venuka
V
Durani
Brandon J
BJ
Sullivan
Thomas J
TJ
Magliery
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases