US 20070298490A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0298490 A1 Sweigard et al. (43) Pub. Date: Dec. 27, 2007 (54) THERMOPHILIC HYDROPHOBIN Related U.S. Application Data PROTEINS AND APPLICATIONS FOR SURFACE MODIFICATION (62) Division of application No. 10/920,876, filed on Aug. 18, 2004, now Pat. No. 7,241,734. (76) Inventors: James A. Sweigard, Elkton, MD (US); Publication Classification Barry Stieglitz, Wynnewood, PA (US) (51) Int. C. C07K I4/00 (2006.01) C7H 2L/04 (2006.01) Correspondence Address: CI2N IS/63 (2006.01) E I DUPONT DE NEMOURS AND (52) U.S. Cl. ...................... 435/320.1; 530/350; 530/371; COMPANY 536/24.3 LEGAL PATENT RECORDS CENTER BARLEY MILL PLAZA 25/1128 (57) ABSTRACT 4417 LANCASTER PIKE WILMINGTON, DE 19805 (US) The present invention relates to a thermophilic hydrophobin, TT1, or a protein or polypeptide Substantially similar thereto, derived from the thermophilic fungus Talaromyces (21) Appl. No.: 11/788,351 thermophilus. The invention further relates to a polynucle otide encoding Such hydrophobin, as well as to materials (22) Filed: Apr. 19, 2007 coated with such hydrophobin. 65 60 55 -0- 3 Hr - - - - - 6 Hr WCA 50 45 - - -A - 16 Hr 40 35 30 25°C 40°C 60°C 80°C Temperoture Patent Application Publication Dec. 27, 2007 Sheet 1 of 3 US 2007/0298490 A1 II:ONTIÒGISITISOTV??INTOIIGNssav?N 305?vºvoaALAGOOAJAH?ãOhashvixos?ownariON™OOTTODITIOvIGÐIVI?LidovxsAAN55vaoNo ?Ô55VIHOX5?ÒNIISSIOTTIGANTIS5xdriology |013 Patent Application Publication Dec. 27, 2007 Sheet 3 of 3 US 2007/0298490 A1 -IH9-º- -IH9--+---- uH9||—-v---- •07009300.089009J 9un?DuÐGUÐ| 9."9|- US 2007/0298490 A1 Dec. 27, 2007 THERMOPHILIC HYDROPHOBIN PROTEINS AND hydrophilic properties, allow them to interact with a wide APPLICATIONS FOR SURFACE MODIFICATION range of material Surfaces. In general, upon contact with either hydrophobic or hydrophilic surfaces, hydrophobin FIELD OF THE INVENTION monomers self-assemble to form a film covering the Surface. A consequence of hydrophobin film formation is a change in 0001. This invention is in the field of biotechnology. surface wettability. Thus on hydrophobic surfaces such as More specifically, this invention pertains to a thermophilic Teflon R film, a hydrophobin coating can increase hydrophi hydrophobin protein and the encoding nucleic acid frag licity as measured by a decrease in the water contact angle. ment, as well as its use as a Surface coating of materials to Conversely, on hydrophilic Surfaces hydrophobin coatings provide beneficial properties. can decrease wettability, measured by an increase in the water contact angle. Particularly Class I hydrophobin SC3 BACKGROUND OF THE INVENTION from the fungus Schizophyllum commune and Class II hydrophobins HFBI and HFBII from Trichoderma reesei 0002 Hydrophobins are small, secreted cysteine-rich have been used in coating studies. HFBI and HFBII formed amphipathic proteins found in fungi. Mature hydrophobin weakly ordered and highly crystalline coatings, respectively, proteins, with secretion signal peptides removed, are gen on water Surfaces (Sermaa, R. et al. Appl. Crystallography erally small proteins that are around 100 amino acids in length. Hydrophobins are characterized by having a pattern 36:499-502). Properties of different hydrophobins, even of 8 highly conserved cysteines that are positioned within among hydrophobins within the same Class, can be quite the mature protein in a 1-2-1-1-2-1 pattern (dashes represent variable in different coating situations. variable numbers of amino acids). Pairs of these 8 cysteines 0005 The novel properties of hydrophobins, including form disulfide bonds, resulting in 4 disulfide bonds that are the ability to self-assemble at interfaces in aqueous Solution necessary for the proper folding of the protein. Though the and under mild conditions, immediately suggest potential 8 cysteines are highly conserved, different hydrophobins are applications for these surface active proteins. Some potential quite variable in their amino acid sequences, some having as applications presented in the literature include: little as 11 % or less sequence identity (Wessels, J. G. H. 0006 use in tissue engineering, particularly in coating of 1994 Annu. Rev. Phytopathol. 32:413-37). Currently, over unnatural Surfaces with a natural protein to increase the 50 hydrophobin sequences from a range of fungal species unnatural Surface biocompatability, e.g., medical implants are known, and are available, for example, through Gen Bank and the National Center for Biotechnology Informa and surgical instruments (Wessels, J. G. H. 1997 Adv. tion (NCBI; Bethesda, Md.). Many fungal species have Microbiol. Physiol. 38:1-45). several reported hydrophobins. Based on differences in 0007 use in drug delivery, especially the delivery of protein primary structure, hydrophobins can be grouped into hydrophilic drugs. Drug oil vesicles coated with a hydro two distinct groups, Class I and Class II. phobin would permit attachment of targeting antibodies to 0003 Genetic studies have implicated hydrophobins as the outside of these vesicles (Wessels, J. G. H. 1997 Adv. playing roles in providing fungal Surface properties. Some Microbiol. Physiol. 38:1-45). hydrophobins, belonging to Class I, are known to form 0008 use in the formation of stable foams in food manu rodlet protein layers found on fungal Surfaces. Rodlet layers facturing and as a natural Surface-active agent in hair prod are extremely hydrophobic and are responsible for generat ucts (Wessels, J. G. H. 1997 Adv. Microbiol. Physiol. 38:1- ing fungal Surface hydrophobicity. Thus all fungal Surfaces 45, Kershaw, M. J. J. Talbot. 1998 Fung. Genet. Biol. 29: including aerial hyphae, fruiting bodies and vegetative 18-33). spores, e.g., conidia, are coated with the hydrophobic domain of self-assembled hydrophobins exposed to the 0009 use to pattern different molecules on a surface with environment and the hydrophilic domain attached or inter nanometer accuracy (Scholtmeijer, K., et al. 2001 Appl. acting with the hydrophilic cell wall. Thus some roles of Microbiol. Biotechnol. 56: 1-8). hydrophobins in nature are to allow fungal hyphae to escape 0010) use to bind or immobilize factors on a hydro into the air from its aqueous environment and search for new phobic support, Such as yeast cells expressing a hydro food sources and Surfaces, to line the internal air spaces in phobin in its cell wall (Nakari-Setala, T., et al. 2002 Schizophyllum commune fruiting bodies in order to prevent Appl. Environ. Microbiol. 68:3385-3391), and several flooding by water of these air channels, and to promote commercially available lipases (Palomo, J. M., et al. interactions between the fungus and the plant during patho 2003 Biomacromol. 4:204-210). genicity, either acting as elicitors of plant defense responses or as stealth factors protecting the invading fungus from 0011 use to develop novel diagnostic sensors through detection and rejection by the plant (Whiteford, J. R. P. D. hydrophobin self-assembly on electrode surfaces (Bilewicz, Spanu 2002 Molec. Plant Pathol. 3: 391-400). R., et al. 2002 J. Phys. Chem. B. 105: 9772-9777). 0004) The best characterized Class I hydrophobin, SC3, 0012 Hydrophobins used in previous studies have gen which is obtained from the fungus Schizophyllum commune, erally been isolated from the natural fungus in which the has been used to study hydrophobin assembly on Surfaces hydrophobin was identified. However, the amounts of pro and the resulting changes in Surface properties. Two Class II tein that can be obtained using this method of preparation are hydrophobins, HFBI and HFBII, isolated from Trichoderma inadequate for commercial applications. A Successful reesei, have also been studied. These hydrophobins have recombinant DNA technology method would be required to been found to be useful as surface coatings to alter the Supply adequate amounts of hydrophobin proteins for com properties of the coated Surfaces. The amphipathic proper mercial uses. An example of recombinant hydrophobin ties of hydrophobins, meaning having both hydrophobic and expression is in WO 00/058342, where the endogenous US 2007/0298490 A1 Dec. 27, 2007 HFBI hydrophobin was overexpressed in Trichoderma 0019. In a third embodiment, the present invention con reesei. Using a recombinant DNA technology method also cerns a recombinant DNA construct comprising any of the allows the creation of hydrophobin variants that may pro isolated polynucleotides of the present invention operably vide Superior properties for use in specific applications. linked to a secretion signal peptide coding sequence and at 0013 Methods of treating surfaces with hydrophobins to least one regulatory sequence. provide a stable coating have been described. In 0020. In a fourth embodiment, the present invention US2003.0134042, Teflon(R) materials were incubated in an concerns an isolated thermophillic hydrophobin polypep SC3 hydrophobin solution at 25° C. and then a heat treat tide, wherein the polypeptide has an amino acid sequence of ment was applied. Aheat treatment of 63°C. or 70° C. in the at least 80%, 85%, 90%, or 95% identity, based on the presence of different detergents at a concentration of 0.1% Clustal W method of alignment, when compared to SEQID was required to obtain strong binding of the hydrophobin to NO:1. The polypeptide most preferably comprises SEQ ID the Teflon R surface. Heat treatments at lower temperatures, NO:1. but above 30°C., in the presence of detergent were partially effective in transitioning the SC3 hydrdphobin to a beta 0021. In a fifth embodiment, the present invention sheet state deemed necessary for strong binding to hydro includes a composite structure comprising a polymer mate phobic Surfaces. rial having a thermophilic hydrophobin coating. 0014) Another method of treating surfaces requires pre 0022. In a sixth embodiment, the present invention pro treatment of the hydrophobin prior to application. In vides a method of treating the surface of an object with a US200301 13454 the disulfide bonds of the SC3 hydro hydrophobin that is performed at a temperature in the range phobin were disrupted by treating with sulphite to add sulfite of greater than 25° C.