Use of single-cell analysis and recombinant protein - GFP fusions to optimise recombinant protein production

Single cell analysis using flow cytometry offers the ability to rapidly optimise many stages of the production of recombinant proteins in bacterial hosts. Physiology can be monitored using membrane-responsive dyes, allowing rapid determination of bacterial health and detection of live, injured and dead subpopulations in shakeflasks and bioreactors, including at high cell density. In addition, unlike conventional analytical tools such as colony forming unit analysis, flow cytometry allows viable non-culturable (VNBC) bacteria to be enumerated and monitored. This phenotype is especially prevalent during recombinant protein fermentations where stress induces a lack of culturability in subpopulations of bacteria.

The fusion of green fluorescent protein (GFP) onto recombinant proteins extends the utility of flow cytometry by permitting analysis of not only the quantity of recombinant protein generated per bacterium, but also estimation of protein folding quality. These parameters can be combined with determination of bacterial viability, and permit comprehensive characterisation of subpopulations within bacterial fermentations. Inclusion body formation can also be analysed on a single-cell level. These analytical methods have been applied to several stages of the production of recombinant proteins, from selection of colonies on agar plates to intensified high cell density cultures in bioreactors, and with a variety of recombinant proteins of varying solubility and difficulty of production in E. coli. The advantages of single cell methods for fermentation development in each of these scenarios will be discussed.