Rosetta-gami B From Novagen

Protein expression in bacteria is a widely used tool to produce important materials for research. The bacterial strain Rosetta-gami B from Novagen combines a number of useful features and mutations, which enhance the yield of eukaryotic proteins in E. coli to a remarkable extent. It combines features of the strains BL21 and its Tuner™ derivative, as well as the Novagen strains Origami™ and Rosetta™, to offer most of the desired mutations which enhance both the expression of eukaryotic proteins and the formation of target protein disulfide bonds in the bacterial cytoplasm.

The host background strain is BL21, which has the advantage of being deficient in two proteases lon (an ATP-dependent protease, which normally degrades abnormal polypeptides) and ompT (an outer membrane protease). The formation of disulfide bonds in the cytoplasm is enhanced by mutations in thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which are selectable on kanamycin and tetracycline, respectively. Due to the lack of these two enzymes, trxB and gor, protein expression does not need to be directed into the periplasm as an oxidative compartment; instead, the respective proteins can be isolated directly from the cytoplasm increasing the overall yield. In addition, the Rosetta-gami B strain supplies six different tRNAs (GGA, AUA, CUA, CGG, AGA, AGG), which are encoded on a plasmid carrying chloramphenicol resistance as selectable marker, to enable efficient translation of foreign transcripts that could otherwise be limited by the different codon usage of E. coli. These six additional tRNA genes are driven by their native promoters. IPTG induced protein expression is widely used to overexpress the protein of interest in bacteria. For this purpose Rosetta-gami B carries another mutation in the lac permease (lacY). This mutation allows uniform entry of IPTG into all cells in the population producing a concentration-dependent, homogeneous level of induction. By adjusting the concentration of IPTG, expression can be regulated from low expression levels up to the robust, fully induced expression levels. Lower level expression may enhance the solubility and activity of proteins that are typically difficult to express.

I have started using this strain for the expression of Fab proteins and I am impressed by the protein yield I am getting from Rosetta-gami B. From a 1000 ml bacterial culture, I get 80-100 µg of purified protein when I use this strain. A typical experiment for protein induction is as follows: I start my pre-culture early in the day and then I inoculate 600 ml medium in shaker bottles with 0.5 ml from this pre-culture and let them grow for about 18 h before I induce the protein expression by adding IPTG. This way, they are well grown but not in the stationary phase. After 4 to 5 h induction with IPTG, the bacteria are harvested by centrifugation. The cell pellet is then dissolved in extraction buffer containing lysozyme and incubated for 30 minutes at room temperature. After that, the solution can be kept frozen until further protein purification processes depending on the procedures used for that particular protein.