ABSTRACT Human neurturin (NTN) is a cystine knot growth factor with potential therapeutic use in diseases such as Parkinson's and diabetes. Scalable high titer production of native NTN is particularly challenging because of the cystine knot structure which consists of an embedded ring comprised of at least three disulfide bonds. We sought to pursue enhanced scalable production of NTN in Escherichia coli . Our initial efforts focused on codon optimization of the first two codons following AUG, but these studies resulted in only a marginal increase in NTN expression. Therefore, we pursued an alternative strategy of using a bicistronic vector for NTN expression designed to reduce mRNA secondary structure to achieve increased ribosome binding and re‐initiation. The first cistron was designed to prevent sequestration of the translation initiation region in a secondary conformation. The second cistron, which contained the NTN coding sequence itself, was engineered to disrupt double bonded base pairs and destabilize the secondary structure for ribosome re‐initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using the bicistronic vector had an additive effect resulting in significantly increased NTN expression. Our strain selection studies were conducted in a miniaturized bioreactor. An optimized strain was selected and scaled up to a 100 L fermentor, which yielded an inclusion body titer of 2 g/L. The inclusion bodies were refolded to yield active NTN. We believe that our strategy is applicable to other candidate proteins that are difficult‐to‐express due to stable mRNA secondary structures. Biotechnol. Bioeng. 2017;114: 1753–1761. © 2017 Wiley Periodicals, Inc.

Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. Here, we have developed a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the cell-free synthesis of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high-throughput prediction of mAb expression ranking to accelerate design build test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.