Journal of Biotechnology 128 (2007) 258–267 Transcript stabilization by mRNA sequences from hrpA of Pseudomonas syringae Elina Hienonen a , Martin Romantschuk b , Fred Fenel c , Suvi Taira a,∗ a Department of Biosciences, Division of General Microbiology, University of Helsinki, Viikki Biocenter, P.O. Box 56, FI-00014, Finland b Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FI-15140 Lahti, Finland c Carbozyme Ltd., Keilaranta 16, FI-02150 Espoo, Finland Received 7 July 2006; received in revised form 11 September 2006; accepted 10 October 2006 Abstract Production of heterologous proteins in bacteria is one of the main applications of biotechnology. Although several high- efficiency expression systems have been developed, different steps in protein production may become rate-limiting depending on the production system and the protein being produced. One bottle neck can be the instability of the mRNA. We have used fragments of the unusually long-living mRNA hrpA from the plant pathogenic bacteria Pseudomonas syringae pathovars tomato and phaseolicola to increase the half-lives of heterologous transcripts. The stabilizing effect was extended to Escherichia coli, as half-lives of several heterologous transcripts were increased from a few minutes to up to 19 min. Production of heterologous proteins was also increased manifold by the addition of the stabilizing hrpA elements. We have mapped the regions of the hrpA transcript necessary and sufficient for the stabilization process. © 2006 Elsevier B.V. All rights reserved. Keywords: mRNA stability; Protein production; hrpA 1. Introduction Microbes are used for the production of foreign proteins for medical and industrial use. Problems still exist in the optimization of the production processes. The main factors affecting the yield of the protein product are promoter activity, transcript stability, ∗ Corresponding author. Tel.: +358 9 19159213; fax: +358 9 19159262. E-mail address: Suvi.Taira@Helsinki.fi (S. Taira). translational efficacy and protein stability, compart- mentalization and conformation. Overproduction of heterologous proteins can also lead to destabilization of the transcripts coding for them (Carrier and Keasling, 1997a,b). Cell growth can be slowed down when the protein is being overproduced, and gene expression can be reduced. Improving the production of heterologous proteins by removing rare codons can also have the opposite results to those intended. mRNA stability can be reduced if the ribosome protection effect offered by the rare codons in the transcript is lost 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.10.010