The cost-effective, easy-to-use T7 expression system has been integral to Escherichia coli protein production since its introduction in 1986. Now, a just-developed enhancement is making it even better by improving yields for myriad recombinant proteins, including enhanced green fluorescent protein (eGFP) and human cytochrome c.
A team led by James Bosco, PhD, research associate, University of Montana and founder of Arktos Life Sciences, recently detailed an auto-expression medium to maximize protein production. Dubbed Bosco Broth, it uses galactose rather than lactose to induce auto-expression.
In experiments, the team reports Bosco Broth “increased yields for routinely-expressed proteins, improved expression of human cytochrome c within a dual expression system,” and achieved “robust auto-expression in lacZ-deficient strains producing proteins with challenging disulfide bonds.” It also showed a “reproducible, 8-fold increase in Streptococcus pyogenes Cas9 (SpCas9) yields at 95% or greater purity.”
When coupled with Arktos’ proprietary production platform, “Bosco Broth…has demonstrated efficiency improvements exceeding 20-fold—often close to 50-fold—relative to industry standards,” Bosco tells GEN.
The research compared Bosco Broth’s ability to produce proteins against four other leading broths, using in-cell fluorescence to measure protein production. Bosco broth had a higher total protein yield—approximately 175 mg/L versus 40 mg/L or less for the other four broths—because it supports a high-density E. coli cell culture and folds efficiently.
Dual-expression capabilities
To test Bosco Broth’s capabilities in a dual expression system, Bosco’s team chose the co-expression model human cytochrome c (Hu-Cytc) and the yeast heme lyase, which are both encoded by the same plasmid. It was completely successful at expressing the protein of interest. (Traditional media is only 50–60% successful, the team pointed out.) Additionally, three times more Hu-Cytc was produced using Bosco broth than when using standard expression protocols. Enzyme activity and functionality were comparable. The team attributed such high productivity to the “greater number of cells engaged in protein production, and an unbiased expression of both proteins of interest.”
Bosco broth also produced disulfide-bonded proteins of interest using the SHuffle T7 Express E. coli. There, galactose was as efficient as IPTG in inducing eGFP.
“This is intriguing because the genetics in SHuffle T7 bacterial strains don’t allow for lactose to work, but galactose does, relative to autoinduction,” Bosco says. “It’s a curious finding, given that there is no evidence that galactose can moonlight in place of lactose in the cellular pathways that oversee lactose metabolism. To my knowledge—and many other folks’—this has never been reported anywhere in the literature.”
Bosco Broth’s key features are its ability to support high-density bacterial cell cultures and produce protein at levels that exceed industry standards. Overall, the scientists consider it a more economical and more versatile option than lactose.
Arktos is finalizing an exclusive licensing agreement from the University of Montana to begin using this method commercially. Further research is being planned to identify the mechanism of action that leads to higher soluble protein output.