A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coli.

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.

Effect of CO2 on uninfected Sf-9 cell growth and metabolism.

A problem in the mass production of recombinant proteins and biopesticides using insect cell culture is CO2 accumulation. This research investigated the effect of elevated CO2 concentration on insect cell growth and metabolism. Spodoptera frugiperda Sf-9 insect cells were grown at 20% air saturation, 27(°) C, and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183, and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4, and 69.3 ± 35.9 h (n = 3 or 4, 95% confidence level), respectively. The viability of cells in all experiments was above 90%, i.e., while increased CO2 concentrations inhibited cell growth, it did not affect cell viability. The osmolality for all bioreactor experiments was observed to be 300-360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not significantly alter the cell specific glucose consumption rate (2.5-3.2 × 10(-17) mol/cell s), but slightly increased the specific lactate production rate from -3.0 × 10(-19) to 10.2 × 10(-19) mol/cell s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:465-469, 2016.