Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins.

High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.

Sense and nonsense from a systems biology approach to microbial recombinant protein production.

The 'Holy Grail' of recombinant protein production remains the availability of generic protocols and hosts for the production of even the most difficult target products. The present review provides first an explanation why the shock imposed on bacteria using a standard induction protocol not only arrests growth, but also decreases the number of colony-forming units by several orders of magnitude. Particular emphasis is placed on findings of numerous genome-wide transcriptomic studies that highlight cellular stress, in which the general stress, heat-shock and stringent responses are the underlying basis for the manifestation of the deterioration of cell physiology. We then review common approaches used to solve bottlenecks in protein folding and post-translational modification that result in recombinant protein deposition in cytoplasmic inclusion bodies. Finally, we suggest a generic approach to process design that minimizes stress on the production host and a strategy for isolating improved hosts.

Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins.

A C-terminal green fluorescent protein (GFP) fusion to a model target protein, Escherichia coli CheY, was exploited both as a reporter of the accumulation of soluble recombinant protein, and to develop a generic approach to optimize protein yields. The rapid accumulation of CheY∷GFP expressed from a pET20 vector under the control of an isopropyl-ß-d-thiogalactoside (IPTG)-inducible T7 RNA polymerase resulted not only in the well-documented growth arrest but also loss of culturability and overgrowth of the productive population using plasmid-deficient bacteria. The highest yields of soluble CheY∷GFP as judged from the fluorescence levels were achieved using very low concentrations of IPTG, which avoid growth arrest and loss of culturability postinduction. Optimal product yields were obtained with 8 µM IPTG, a concentration so low that insufficient T7 RNA polymerase accumulated to be detectable by Western blot analysis. The improved protocol was shown to be suitable for process scale-up and intensification. It is also applicable to the accumulation of an untagged heterologous protein, cytochrome c(2) from Neisseria gonorrhoeae, which requires both secretion and extensive post-translational modification.

Recombinant protein production: a comparative view on host physiology.

The fifth meeting organised by the EFB on the influence of host physiology on recombinant protein production was held in Alghero, Sardinia in September 2008. Conclusions from the meeting included that a very wide range of hosts are still needed to produce recombinant proteins of quality adequate for use in human healthcare. CHO cells can produce excellent titres, but it remains impossible to predict how a transfection line will perform, especially at high cell density when productivity declines: Pichia pastoris might replace mammalian cell cultures for many applications. A series of transcriptomic and proteomic studies have generated large datasets that provide a valuable resource for understanding how to improve recombinant protein production, but this remains a promise rather than a fait accompli. E. coli remains the workhorse for over half of the recombinant proteins produced by the biopharmaceutical industries. Until recently, its Achilles heel was its inability to glycosylate eukaryotic proteins, a problem for which the development of bacterial N-linked protein glycosylation systems offers an imminent solution.