(643a) Large-Scale Production of Elastin-Like Polypeptide-Tagged Organophosphate Hydrolase (OPH)

Elastin-like polypeptides (ELPs) are an attractive, non-chromatographic alternative to conventional tags for protein purification. They are comprised of repeating pentapeptides that selectively and reversibly aggregate in response to shifts in osmostic strength and temperature. These ELPs can be used in conjunction with self-cleaving inteins to form economical, protease-free purification tags. However, some limitations of the tags exist. First, rapid high-level expression of the tagged target is difficult with conventional strategies and strains, presumably due to the metabolic arising from expression of the highly repetitive ELP sequence. Therefore, ELP-tagged recombinant proteins are typically expressed in shake flask cultures at low temperatures without induction for 24-48 hours, where protein production relies on leaky expression from a given promoter (usually T7). However, long induction times lead to the additional problem of intein premature cleavage. Further, low oxygen levels have been correlated to increased premature intein cleavage in some cases as well.

High cell-density fermentations are important for large scale production of recombinant proteins in general, including those tagged with self-cleaving ELP. Thus, the development of effective fermentation strategies for these tags is an important goal for the future of this technology, where the specific demands of the ELP tag provide unique challenges. In this paper we report an optimized high cell-density fermentation strategy for the production of ELP-tagged organophosphate hydrolase (OPH). Specifically, we investigated the protein yields attainable with leaky expression from the T7 promoter at high cell densities in both rich and defined media. We also examined the effect of dissolved oxygen on intein premature cleaving. These results thus also provide an interesting case study for the production of a highly metabolic-burdensome protein in fermentation cultures.