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The minimum requirement for unsaturated fatty acids was investigated in E. coli using a mutant impaired in the synthesis of vaccenic acid. Exogenously supplied palmitic acid was incorporated by this mutant which led to a reduction in the proportion of cellular unsaturated fatty acids. Growth was impaired as the level of saturated fatty acids approached 76% at 37 degree C and 60% at 30 degree C. The basis of this growth inhibition was investigated. Most transport systems and enzymes examined remained active in palmitate-grown cells although the specific activities of glutamate uptake and succinic dehydrogenase were depressed 50%. Fluorescent probes of membrane organization indicated that fluidity decreased with palmitate incorporation. Temperature scans with parinaric acid indicated that rigid lipid domains exist in palmitate-grown cells at their respective growth temperature. Freeze-fracture electron microscopy confirmed the presence of phase separations (particle-free areas) in palmitate-grown cells held at their growth temperature prior to quenching. The extent of this separation into particle-free and particle-enriched domains was equivalent to that induced by a shift to 0 degree C in control cells. The incorporation of palmitate increased nucleotide leakage over threefold. The cytoplasmic enzyme beta-galactosidase was released into the surrounding medium as the concentration of unsaturated fatty acid approached the minimum for a particular growth temperature. Lysis was observed as a decrease in turbidity when cells which had been grown with palmitate were shifted a lower growth temperature. From these results we propose that leakage and partial lysis are the major factors contributing to the apparent decrease in growth rate caused by the excessive incorporation of palmitate. Further, we propose that membrane integrity may determine the minimum requirement for unsaturated fatty acids in E. coli rather than a specific effect on membrane transport and/or membrane-bound enzymes.

We have developed a Threshold System immunoligand assay for the quantitation of residual, process-specific, Escherichia coli host cell contaminant proteins (HCP) in somavubove (a normal sequence recombinant bovine somatotropin). The assay has a dynamic range of 2 to 160 ng/mL, with a limit of quantitation of 2 ng/mL. Daily analytical precision (CV) for six replicates of the designated somavubove laboratory standard is 3.0%. Cumulative analytical precision for multiple assay runs of this standard (a total of 69 laboratory standard replicates) is 8.4%. A conservative alert limit of 100 ng/mL has been assigned in order to assure analytical precision for each assay sample under conditions of stoichiometric antibody excess. Although the assay was designed for use as a profile-release specification assay, it has also been used to validate removal of HCP by the proprietary somavubove purification process. This use is consistent with regulatory guidelines related to "well characterized" recombinant biopharmaceutical proteins.

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