Several years ago we tried in vitro expression and were successful only at the level of seeing bands on a silver-stained gel when we wanted big Coomassie bands that indicated expression levels suitable for structural studies. We would wish to mix and match constructs with in vitro methods to get multiple types of complexes without separately cloning multiple components.
For proteins in the 100Kd size what is the typical achievable expression level in various systems currently?
It depends on what kind of proteins you are interested in. You should think about whether you would like to have post translational modifications or not. If no e.coli expression should be sufficient. If you are interested in a eukaryotic protein complex you should at least go for yeast or better insect cells. The gold standard though the yields might be lower is to work with a suspension cell line like 293F (Freestyle System Invitrogen).
Cell Free Sciences has an optimized system that works great in my friend's hands: ttp://www.cfsciences.com/eg/PremiumKit.html. Good luck!
An alternative strategy might be to use Agrobacterium mediated transient gene expression in tobacco leaves. It supports co-expression of several transgenes by simply mixing the Agrobacteria carrying the corresponding vectors. The system is suitable for both analytical purposes as well as for production of gram amounts of recombinant protein. Different signals can be used to target to proteins to different subcellular compartments to optimize accumulation and control PTMs. Nicotiana tabacum and Nicotiana benthamiana plants are easy and cheap to grow, especially if a greenhouse or phytochamber can be accessed. Expression (or rather protein accumulation) levels are highly protein dependend as the cells have quality control systems (protein folding and degradation) and can range from minute amounts up to a milligram of recombinant protein per gram of wet leaf tissue.
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