The phrase “protein dependent” is ubiquitous among structural biologists, reflecting the inherent variability encountered when working with different protein sequences. In practice, each protein often requires distinct conditions for successful in vitro reconstitution, and different experimental assays may demand unique affinity or solubility tags. Consequently, researchers must often generate a large number of expression vectors for any given target, a process that can be both time-consuming and costly.
Here, we present a “one fragment, many solutions” cloning system that enables a single DNA fragment to be efficiently inserted into a suite of 35 versatile expression vectors. This collection includes constructs with solubility enhancers, fluorescent reporters, and high-affinity purification tags such as Strep, AviTag, and FLAG. The vectors also incorporate multiple antibiotic resistance genes to support co-expression. They also span both bacterial and mammalian backbones, facilitating rapid assessment of protein expression levels and quality across systems.
Importantly, all vectors utilise a ccdB-based Golden Gate cloning system with a conserved 3’ and 5’ overhang. This virtually eliminates background colonies and allows efficient assembly in a 1 µL reaction at 37°C for just 30 minutes. This streamlined framework substantially reduces the time and cost associated with vector generation, accelerating the path from gene to functional protein.