Abstract
This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.
Highlights
Secretion of heterologous proteins into the culture medium is an often-used tool in biotechnology
MtrF is a suitable exporter for protein secretion The aim of this study was to establish outer membrane cytochromes of S. oneidensis as transporters for export of proteins into the medium using the type II secretion pathway
Previous work revealed that of the five putative outer membrane cytochromes encoded in the genome of S. oneidensis only three (OmcA, MtrC and MtrF) are exposed to the cell surface [8] and are potential targets for the Determination of the MtrF export signal The protein structure defining T2SS substrates like MtrF for translocation through the outer membrane is unknown
Summary
Secretion of heterologous proteins into the culture medium is an often-used tool in biotechnology. This technique is applied in order to reduce the protein purification burden or to engineer cells to thrive on polymeric substrates via the use of excreted hydrolytic enzymes. One has to distinguish between one-step and two-step export machineries. One-step export machineries transport unfolded or partially folded proteins directly from the cytosol to the medium, which requires that the protein has to fold correctly without accessory proteins like foldases or chaperons [18, 28]. It was discovered that special proteins like for example YebF in E. coli are secreted across the outer membrane via an unknown mechanism and that a fusion to these proteins could allow the co-transport of cargo proteins [41]
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