Microbial Cell Factories

Background: Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results: In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.l-1 of purified active LCI. Conclusion: These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Background Escherichia coli is by far the simplest, but one of the most widely used host cell for the production of recombinant proteins [1]. Nevertheless, the efficient translocation across the inner membrane and proper periplasmic folding of eukaryotic proteins stabilized by multiple disulfide bonds remains challenging for this organism [2]. Unfortunately, many proteins of which there is a great biotechnological or biomedical interest are secreted proteins containing essential disulfide bonds for their native structure. Either premature cytoplasmic protein folding or incorrect disulfide bond formation in the bacterial periplasm are two known limitations in the overproduction of secreted proteins [3]. Recently, it has been reported that signal sequences promoting co-translational translocation improved the translocation of heterologous proteins [4]. Therefore, targeting these recombinant precursors to the cotranslational signal recognition particle (SRP) dependent pathway conceivably could result in much higher levels of periplasmic proteins than directing them posttranslationally to the SecYEG translocase [5]. Strategies to overcome folding problems due to disulfide bond formation have primarily focused on the co-production of protein disulfide isomerases [3]. For example, the overproduction of DsbC, a periplasmic thiol isomerase, resulted in large amounts of native human tissue plasminogen activator [6]. Published: 29 October 2009 Microbial Cell Factories 2009, 8:57 doi:10.1186/1475-2859-8-57 Received: 9 September 2009 Accepted: 29 October 2009 This article is available from: http://www.microbialcellfactories. com/content/8/1/57 © 2009 Puertas and Betton; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microbial Cell Factories 2009, 8:57 http://www.microbialcellfactories. com/content/8/1/57