Amino Acid Accumulation Limits the Overexpression of Proteins in Lactococcus lactis
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Authors: | Ravi K. R. Marreddy Eric R. Geertsma Hjalmar P. Permentier Joao P. C. Pinto Jan Kok Bert Poolman |
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Affiliation: | 1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.; 2. Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.;Baylor College of Medicine, United States of America |
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Abstract: | BackgroundUnderstanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media.Methodology/Principal FindingsThe proteomic profiles of cells growing in media in which the proteins were expressed to high or low levels suggested a limitation in the availability of branched-chain amino acids, more specifically a too limited capacity to accumulate these nutrients. By supplying the cells with an alternative path for accumulation of Ile, Leu and/or Val, i.e., a medium supplement of the appropriate dipeptides, or by engineering the transport capacity for branched-chain amino acids, the expression levels could be increased several fold.ConclusionsWe show that the availability of branched chain amino acids is a critical factor for the (over)expression of proteins in L. lactis. The forward engineering of cells for functional protein production required fine-tuning of co-expression of the branched chain amino acid transporter. |
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