E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins
Abstract
The expression and purification of large recombinant proteins or protein complexes is problematic for some
biotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biological
characterization or reach commercialization, when large proteins or protein complexes are expressed in E. coli
via the popular T7-based plasmid-driven expression system.
There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of its
drawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copy
number, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, such
as gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter of
debate.
Plant plastidial NAD kinases (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23) are a classic example of
proteins with high molecular weight, that are difficult to express and purify with traditional T7-based tech-
nology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thaliana
NADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-free
chromosomal modifications.
Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein than
classic T7 expression systems, as well as better production, thus confirming that expression from one single
chromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and applied
research.