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Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

The sensitivity of high-precision liquid chromatography/combustion isotope ratio mass spectrometry (LC/C-IRMS) was improved by more than two orders of magnitude by modifications which permit optimization of flows. The lower limits for high precision are about 3 pg of analyte on-column for flow injection and LC modes. Pneumatic aerosol spray coating was implemented and compared with simple dip coating. The signal enhancement for aerosol compared with dip coating is up fourfold at higher levels but drops to less than twofold at lower levels, while the dynamic range is extended at higher levels by fourfold at high sample load levels. LC separations for underivatized tocopherols, retinyl acetate and ergocalciferol are demonstrated with precision and accuracy of about 6I3C < 2.5% for replicates. This work demonstrates sensitivity useful for a wide range of LC analyses and extends high-precision LC/C-IRMS to the determination of several fat-soluble vitamins.

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