Fungal hydrophobins are a group of surface active, self-assembling proteins. The filamentous fungus Trichoderma reesei produces two (class II) hydrophobins, HFBI and HFBII. We have studied how these water-soluble hydrophobins behave in two-phase systems using a series of nonionic surfactants with different characteristics. It was found that both hydrophobins, but especially HFBI, had a very high affinity for the surfactants. The highest partitioning coefficient, over 2500, was observed for HFBI with C(11)EO(2). Reducing the disulfides in the protein resulted in a complete loss of affinity for the surfactant, which demonstrates that the interaction is dependent on the disulfide-stabilized conformation. The hydrophobins could be efficiently extracted back from the surfactant phase by addition of alcohols such as isobutanol. Effects of the type of surfactant, temperature, pH, and ionic strength were investigated. The use of this method for purifying the proteins from crude fungal culture supernatants is demonstrated and implications of the protein-polymer interaction are discussed.

In this work we describe the new concept of using fungal hydrophobins as efficient tags for purification of recombinant fusion proteins by aqueous two-phase separation. Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Some characteristics of hydrophobins are that they are relatively small (approximately 100 amino acids), they contain eight disulfide-forming Cys residues in a conserved pattern, and they self-assemble on interfaces. The aqueous two-phase systems studied were based on nonionic surfactants that phase-separate at certain temperatures. We show that the use of hydrophobins as tags has many advantages such as high selectivity and good yield and is technically very simple to perform. Fusion proteins with target proteins of different molecular size were compared to the corresponding free proteins using a set of different surfactants. This gave an understanding on which factors influence the separation and what rationale should be used for optimization. This unusually strong and specific interaction between polymeric surfactants and a soluble protein shows promise for new developments in interfacing proteins and nonbiological materials for other applications as well.

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BackgroundActinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known.ResultsHere, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element.ConclusionsThe complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.