An engineered E. coli ribosome with tunable translation rates enhances recombinant protein expression

Using E. coli for the expression of recombinant proteins in their soluble active form continues to be a challenge in biotechnology. Issues including low yields of full‐length, soluble, and functional proteins fundamentally limit discovery, development, and manufacturing of commercially, industrially, and medically relevant proteins. In many cases, improvements in protein solubility have been achieved by codon optimization, over‐expression of chaperone proteins, solubility tags, and by varying expression conditions. Optimization of these parameters is often done iteratively and on a case‐to‐case basis, which can be slow and expensive. To overcome limitations in recombinant protein expression, we report a genomically engineered strain of E. coli with a tunable ribosome enabling modulation in translation rates under isothermic conditions. Additionally, we show that cell extracts from this strain of E. coli can support a cell‐free protein synthesis platform capable of on‐demand production of recombinant proteins. This platform technology enables two key advances. First, we provide quantitative evidence that tuning protein synthesis rates improves soluble fractions of traditionally intractable proteins such as the human MEK1 and IRKβ. In each case, soluble fractions increased from <20% to >80% of total protein when compared with expression in BL21 cell extracts. 2) This platform technology is plug‐and‐play, allowing the user to identify the optimal translation rate condition for a desired protein in a high‐throughput manner, which can then be scaled‐up for desired protein yields. The timescale from DNA template to the optimized protein expression condition is 24–48 hours, notably faster than traditional methods involving cell transformations, and optimization of expression times and temperatures. Additionally, our data suggest that this approach obviates the need for solubility tags, enabling the expression of proteins in their wild‐type forms. Our work provides a new tool for studying protein folding in the context of ribosomal kinetics, and a new platform for manufacturing traditionally difficult proteins. Support or Funding Information This work was supported by DARPA and the the David and Lucille Packard Foundation Fellowship. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .