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An analysis of a proteomic is conducted over time with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-litre scale using two-dimensional polyacrylamide gel electrophoresis. E.coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression.

The cryptic ushA gene (ushA(super.c)) in Salmonella enterica natural isolates (serotype Typhimurium) has been seen to be inactivated by a single missense mutation, the first reported gene inactivation in natural isolates of bacteria via a missense mutation. Results indicate the evolution of new enzymes involving a silent gene which produces an relatively inactive or inactive product. They also indicate evolution of a novel, although unknown, enzyme specificity by one amino acid change.

An investigation of the in vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC was conducted using genetic selections. Results indicated DsbC acts as a disulfide isomerase. Furthermore, DsbA is a more effective oxidant than DsbC of protein dithiols. Proteins which are effected by DsbC deletions do not have cysteines paired consecutively.

A bacterial expressing system helps isolate the microtubule-binding region (MTBR) of the microtubule associated protein (MAP) 2 and site-directed mutagenesis helps study the interactions between microtubules and MAP. The MTBR enhances tubulin polymerization. The binding energies of the three second repeated peptide sequences remain unchanged during MAP binding to microtubules, confirming that the second sequence repeat is vital for microtubule binding.

The oxidized state of dsbA periplasmic protein catalyzes disulfide bond formation in proteins. The dsbA protein refolds the misfolded IGF-I by restructuring two disulfide bonds. Disulfide isomerase is the reduction state of dsbA protein. The function of the dsbA protein depends on the redox state of the protein under physiological conditions.

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high‐throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally‐derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single‐cell to >99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:584–592, 2018 [ABSTRACT FROM AUTHOR]