WO2001074864A1 - Protein capable of self-assembly at a hydrophobic-hydrophilic interface and uses thereof - Google Patents
Protein capable of self-assembly at a hydrophobic-hydrophilic interface and uses thereof Download PDFInfo
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- WO2001074864A1 WO2001074864A1 PCT/NL2001/000268 NL0100268W WO0174864A1 WO 2001074864 A1 WO2001074864 A1 WO 2001074864A1 NL 0100268 W NL0100268 W NL 0100268W WO 0174864 A1 WO0174864 A1 WO 0174864A1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K3/00—Materials not provided for elsewhere
- C09K3/18—Materials not provided for elsewhere for application to surfaces to minimize adherence of ice, mist or water thereto; Thawing or antifreeze materials for application to surfaces
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
Definitions
- the present invention relates to a protein capable of self-assembly at a hydrophobic-hydrophilic interface.
- Hydrophobins are a well-defined class of proteins ( essels et al, 1997) capable of coating a surface, rendering a hydrophobic surface hydrophilic, and often vice versa. They have a conserved sequence
- X represents any amino acid, and n and m, of course, independently represent an integer.
- a hydrophobin has a length of up to 125 amino acids.
- the cysteine residues (C) in the conserved sequence are part of disulphide bridges. These hydrophobins are typically isolated from fungi like Schizoph llum commune .
- the present invention relates to a protein according to the preamble, characterized in that the protein is an at least partially purified protein comprising a polypeptide having at least 40% identity and at least 5% similarity to at least one polypeptide chosen from the group consisting of i) amino acids 29 - 131 of SEQ NO. 1 and ii) amino acids 29 - 133 of SEQ. NO. 2.
- a new class of hydrophobin-like proteins is provided differing markedly from known hydrophobins, in that the above-defined conserved sequence is not present.
- sequences may comprise insertions or deletions .
- similarity used in association with polypeptide denotes conservative substitutions.
- Conservative substitutions are substitutions in which one amino acid is replaced with another, where the following amino acids are considered similar: A,S,T; D,E; N,Q; R,K;
- the protein may be derived from a filamentous bacterium, in particular a bacterium capable of forming aerial hyphae such as an Actinomycete , and more specifically the filamentous bacterium may be a Streptomvces species.
- a Streptomyces species from which the protein may be isolated using standard procedures for the isolation of hydrophobins is a Streptomyces species which has been transformed with a construct that can be isolated from an E ⁇ _ coli strain which has been deposited on 14 March, 2000 under accession number CBS 102638 with the Centraalbureau voor Schimmelcultures (Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, the Netherlands) .
- the identity is at least 50%, more preferably at least 60% and most preferably at least 70%.
- the similarity is at least 7%, preferably at least 10% and more preferably at least 15%.
- the protein comprising said polypeptide may contain any additional sequences, unusual aminoacids, both with respect to stereo-chemistry as to structure, as long as the capability of self-assembly at a hydrophobic-hydrophilic interface is not compromised.
- the present invention also relates to a method of coating a surface of an object, wherein a protein according to the present invention is used.
- the object is chosen from the group consisting of a window, a contact lens, a biosensor, a medical device, a container used for an assay or storage, the hull of a vessel (ship) , or a frame or body- work of a car, and a solid particle, whereby a solution comprising the protein according to the invention is contacted with said object.
- a hydrophilic surface is coated with an amount of protein sufficient to provide the coated surface of the object with a contact angle for water larger than 60°
- a hydrophobic surface is coated with an amount of protein sufficient to provide the coated surface of the object with a contact angle for water smaller than 90°
- the present invention also relates to a method of stabilizing a dispersion, such as an oil or fat in water- dispersion, wherein a protein according to the invention is used to stabilize the dispersed particles.
- the stabilized particles may also be solid particles, such as latex spheres suitable for assays, such as immuno-assays . It is thought that a treatment at a temperature between 30° and 90°C, optionally in the presence of a surfactant and as disclosed in PCT/NL01/00084 , the description of which is incorporated by reference, may induce a conformational change resulting in a substantially irre- versible change in the structure of the protein, as a result of which a coating is rendered insoluble.
- the proteins according to the invention isolated until now by the present inventors have only up to one disulphide bridge. This may be advantageous for particular applications. Should this be desirable or necessary, it is thought that the hydrophobin may be stabilized as disclosed in PCT/NLOl/00082 , the description of which is included by reference.
- the protein according to the invention may be iso- lated, should this be desirable or necessary, using the method disclosed in PCT/NLOl/00083, the description of which is included by reference.
- fig. 1 depicts an SDS-PAA gel showing the protein according to the present invention
- fig. 2 depicts a Northern blot
- fig. 3 shows a picture of an immuno-gold labeling of RdlA and RdlB proteins at the outer surface of aeral hyphae
- fig. 4 shows a picture indicating the difference in attachment of hyphae of wild-type Streptomyces lividans and a control strain (S ⁇ . lividans) lacking RdlA and RdlB proteins to polystyrene, and a graph quantitatively showing this relative binding for both S _ lividans and S ⁇ coelicolor .
- Streptomyces coelicolor A3 strain M145 and Streptomyces lividans TK23 (Kieser et al . , 2000) were used throughout this study, while Streptomyces griseus (DSMZ 40236) and Streptomyces tendae T ⁇ 901/8c (Richter et al . , 1998) were used to establish whether other streptomycetes contain homologues of rdlA and rdlB .
- Vectors are summarised in Table 1.
- Strepto- ⁇ yces strains were grown at 30 °C on solid MS agar medium or in YEME medium as liquid shaken cultures (Kieser et al . , 2000). Spores were harvested with water and stored as described previously (Kieser et al . , 2000) .
- S. coelicolor and S. lividans were grown in 96-well flat-bottomed microtiter plates (Costar, Corning Incorporated, USA) containing 200 ⁇ l NMMP (Kieser et al . , 2000; in the absence of PEG6000 and using 50 mM glucose as a carbon source) .
- spores Prior to inoculation, spores, stored at -20 °C in 20% glycerol, were washed and diluted in medium to a final concentration of 5 • 10 s spores ml "1 . 96 well flat- bottomed microtiter-plates were filled with 200 ⁇ l spore suspension per well .
- the rdl genes were deleted by replacing a 0.8 kb Blpl/Scal fragment of pC46b (see Table 1) containing the entire coding sequence of rdlA and 136 bp of rdlB as well as the interspersed promoter region by a 1.4 kb Smal fragment containing the hygromycin B resistance gene (Zalaca ⁇ n et al . , 1986) . This resulted in vector pC46c.
- a 1.8 kb Smal fragment containing the apramycin resistance gene was cloned in the Xbal site of pC46c to select for double crossover events, resulting in plasmid pC46d.
- proteins were stained with 0.25% Coomassie Brilliant Blue G-250 (CBB) or blotted onto a polyvinylidenedifluoride (PVDF) membrane using semi -dry blotting.
- CBB Coomassie Brilliant Blue G-250
- PVDF polyvinylidenedifluoride
- N-terminal sequencing a PVDF membrane was stained with CBB and a slice of the membrane containing the protein was excised. After destaining with 30% methanol, the N-terminal sequence was determined using a pulse liquid sequenator on line connected to a PTH analyser (Eurosequence, Groningen, The Netherlands) . To determine N-terminal sequences of internal peptides, the protein was eluted from the SDS-PAA gel followed by a tryptic digestion. Peptides were sequenced after separation on a C18 reversed phase HPLC column.
- Antibodies against RdlA and RdlB were raised by injecting a mixture of these proteins of S. lividans eluted from a SDS- PAA gel. PVDF membranes were treated with diluted anti- RdlA/RdlB serum (1:1000) as described (Harlow and Lane, 1988) .
- IMMUNOLOCALIZATION Fixation, embedding and immuno-labelling of cultures were performed as described (W ⁇ sten et al . , 1994) with the modification that K4M was substituted for Unicryl .
- Polyclonal antibodies raised against a mixture of RdlA and RdlB of S. lividans were purified with an acetone powder of mycelium (Harlow et al . , 1988) of a liquid shaken culture and diluted 1000 times.
- Streptomyces lividans grown on solid MS medium were treated with 2% SDS at 100 °C. After washing with water and lyophilizing, this was followed by an extraction with trifluoroacetic acid (TFA) . SDS-PAGE of the SDS-soluble fraction showed a complex pattern of polypeptides (results not shown) . Among the proteins that were insoluble in hot SDS but soluble in TFA, an abundant polypeptide, called Rdl, was observed with an apparent molecular weight of 18 kDa ( Figure 1, left panel).
- Rdl was the main protein that dissolved ( Figure 1, right panel) .
- the extract formed an SDS-insoluble complex upon shaking that could be dissociated with TFA.
- Similar results as obtained with S. lividans were obtained with cultures of S. coelicolor.
- N-terminal sequencing revealed that the Rdl protein of S. lividans running at the 18 kDa position was in fact a mixture of two similar proteins, called RdlA and RdlB, with slightly different N-termini (Seq. 1 starting at amino acid 29 and Seq. 2 starting at amino acid 29) .
- N- termini of two internal peptides were determined that resulted from a tryptic digestion of a mixture of RdlA and RdlB.
- a radioactive degenerated oligonucleotide based on one of the peptides was used to screen a cosmid library of S. coelicolor A3 (2) (Redenbach et al . , 1996).
- the oligonucleotide hybridised to the overlapping cosmids C46 and C61.
- the hybridising fragment of cosmid C46 was contained on a 4.5 kb Sail fragment. This fragment was cloned in pBluescript KS+ in the unique Sail site, and introduced in E. coli DH5a (deposited on 14 March, 2000 under accession number CBS 102638 with Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, The Netherlands) and partially sequenced.
- An ORF was identified that encodes a polypeptide of 131 aa.
- the coding sequences of rdlA and rdlB hybridised to the same unique fragments of genomic DNA of S. coelicolor and S. lividans digested with a variety of enzymes (one at a time) .
- a 4.5 kb Sail fragment hybridised to both rdlA and rdlB .
- a slightly larger genomic fragment hybridised after digestion with Blpl , while digestion with Pstl resulted in a fragment of about 8 kb (data not shown) .
- rdlA AND rdlB ARE EXPRESSED IN AERIAL HYPHAE
- RNA was hybridised with a probe representing the coding sequence of rdlA or rdlB ( Figure 2) . Expression of both rdl genes correlated with the formation of aerial hyphae. rdl mRNA was not detected in 1-day-old cultures (lower panel lane 1) not yet forming aerial hyphae, nor in 3,
- RdlA and RdlB were localized using an antiserum raised against a mixture of RdlA and RdlB of S. lividans .
- immuno-gold-labelling was observed at the outer surface of cell walls of aerial hyphae ( Figure 3) and spores, but not of submerged hyphae (data not shown) .
- the reactive layer at the outside of the wall was sometimes detached, indicating that it is a discrete layer.
- the antiserum did not react with cultures not forming aerial hyphae, i.e. cultures of S. lividans and S. coelicolor grown in liquid shaken medium, and 1-day-old cultures grown on solid medium.
- the genes coding for the protein according to the invention may be introduced into a vector which is used to transform a host suitable for obtaining the protein in good yield and allowing for easy purification with the desired purity. It is anticipated that for hosts excreting the protein according to the present invention even culture medium may be used to coat a surface. By eliminating the protein-producing organism, the supernatant qualifies as containing at least partially purified protein.
- Val lie Asn Leu Val Pro lie Gin Asp lie Asn Val Leu Ala Asp Asp 85 90 95
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Abstract
The invention relates to a hydrophobin-like protein capable of self-assembly at a hydrophobic-hydrophilic interface. The protein according to the present invention belongs to a new class of protein and is an at least partially purified protein comprising a polypeptide having at least 40 % identity and at least 5 % similarity with at least one polypeptide chosen from the group consisting of i) amino acids 29 - 131 of SEQ NO. 1 and ii) amino acids 29 - 133 of SEQ. NO. 2.
Description
PROTEIN CAPABLE OF SELF-ASSEMBLY AT A HYDROPHOBIC-HYDROPHILIC INTERFACE AND USES THEREOF
The present invention relates to a protein capable of self-assembly at a hydrophobic-hydrophilic interface.
Such proteins are known in the art, and in particular hydrophobins have been disclosed. Hydrophobins are a well-defined class of proteins ( essels et al, 1997) capable of coating a surface, rendering a hydrophobic surface hydrophilic, and often vice versa. They have a conserved sequence
Xn-C-X5.9-C-C-X11_39-C-X8_23-C-X5_9-C-C-X6_18-C-Xm X, of course, represents any amino acid, and n and m, of course, independently represent an integer. In general, a hydrophobin has a length of up to 125 amino acids. The cysteine residues (C) in the conserved sequence are part of disulphide bridges. These hydrophobins are typically isolated from fungi like Schizoph llum commune .
It is desirable to have the disposal of a varied range of proteins having the above property to accommodate the requirements for any particular application of said proteins, such as coating objects. Therefore the present invention relates to a protein according to the preamble, characterized in that the protein is an at least partially purified protein comprising a polypeptide having at least 40% identity and at least 5% similarity to at least one polypeptide chosen from the group consisting of i) amino acids 29 - 131 of SEQ NO. 1 and ii) amino acids 29 - 133 of SEQ. NO. 2.
Thus, according to the present invention, a new class of hydrophobin-like proteins is provided differing markedly from known hydrophobins, in that the above-defined conserved sequence is not present.
In the present application, the term "identity" used in association with polypeptide is defined, in accordance with the state of the art, as having exactly matched amino acid residues. Here, sequences may comprise insertions or
deletions .
The term "similarity" used in association with polypeptide denotes conservative substitutions. Conservative substitutions are substitutions in which one amino acid is replaced with another, where the following amino acids are considered similar: A,S,T; D,E; N,Q; R,K;
I,L,M,V; F,Y,W.
The protein may be derived from a filamentous bacterium, in particular a bacterium capable of forming aerial hyphae such as an Actinomycete , and more specifically the filamentous bacterium may be a Streptomvces species. A Streptomyces species from which the protein may be isolated using standard procedures for the isolation of hydrophobins, is a Streptomyces species which has been transformed with a construct that can be isolated from an E^_ coli strain which has been deposited on 14 March, 2000 under accession number CBS 102638 with the Centraalbureau voor Schimmelcultures (Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, the Netherlands) . Preferably, the identity is at least 50%, more preferably at least 60% and most preferably at least 70%.
Also, the similarity is at least 7%, preferably at least 10% and more preferably at least 15%.
It is thought that such proteins more specifically define proteins having better suitability for coating a surface. It goes without saying that, provided the polypeptide as defined above is present, the protein comprising said polypeptide may contain any additional sequences, unusual aminoacids, both with respect to stereo-chemistry as to structure, as long as the capability of self-assembly at a hydrophobic-hydrophilic interface is not compromised.
In accordance with the above, the present invention also relates to a method of coating a surface of an object, wherein a protein according to the present invention is used.
According to a preferred embodiment, the object is chosen from the group consisting of a window, a contact lens, a biosensor, a medical device, a container used for an assay or storage, the hull of a vessel (ship) , or a frame or body- work of a car, and a solid particle, whereby a solution comprising the protein according to the invention is contacted with said object.
If it is desired to change the hydrophobic/hydrophilic nature of a surface, a hydrophilic surface is coated with an amount of protein sufficient to provide the coated surface of the object with a contact angle for water larger than 60°, and a hydrophobic surface is coated with an amount of protein sufficient to provide the coated surface of the object with a contact angle for water smaller than 90° . It is also possible to use the coated surface to chemically attach various compounds, such as prosthetic groups, antibodies etc., to the protein according to the invention using methods well-known in the art.
The present invention also relates to a method of stabilizing a dispersion, such as an oil or fat in water- dispersion, wherein a protein according to the invention is used to stabilize the dispersed particles. The stabilized particles may also be solid particles, such as latex spheres suitable for assays, such as immuno-assays . It is thought that a treatment at a temperature between 30° and 90°C, optionally in the presence of a surfactant and as disclosed in PCT/NL01/00084 , the description of which is incorporated by reference, may induce a conformational change resulting in a substantially irre- versible change in the structure of the protein, as a result of which a coating is rendered insoluble.
In contrast to known hydrophobins, the proteins according to the invention isolated until now by the present inventors, have only up to one disulphide bridge. This may be advantageous for particular applications. Should this be desirable or necessary, it is thought that the hydrophobin may be stabilized as disclosed in PCT/NLOl/00082 , the description of which is included by reference.
The protein according to the invention may be iso-
lated, should this be desirable or necessary, using the method disclosed in PCT/NLOl/00083, the description of which is included by reference.
The invention will now be illustrated with reference to the following examples and the drawing in which fig. 1 depicts an SDS-PAA gel showing the protein according to the present invention; fig. 2 depicts a Northern blot; fig. 3 shows a picture of an immuno-gold labeling of RdlA and RdlB proteins at the outer surface of aeral hyphae; and fig. 4 shows a picture indicating the difference in attachment of hyphae of wild-type Streptomyces lividans and a control strain (S^. lividans) lacking RdlA and RdlB proteins to polystyrene, and a graph quantitatively showing this relative binding for both S _ lividans and S^ coelicolor .
PREPARATIONS
METHODS Strains and plasmids
Cloning in Escherichia coli was done in DH5c. or JM110. Streptomyces coelicolor A3 (2) strain M145 and Streptomyces lividans TK23 (Kieser et al . , 2000) were used throughout this study, while Streptomyces griseus (DSMZ 40236) and Streptomyces tendae Tύ 901/8c (Richter et al . , 1998) were used to establish whether other streptomycetes contain homologues of rdlA and rdlB . Vectors are summarised in Table 1.
GROWTH CONDITIONS AND MEDIA
Strepto-πyces strains were grown at 30 °C on solid MS agar medium or in YEME medium as liquid shaken cultures (Kieser et al . , 2000). Spores were harvested with water and stored as described previously (Kieser et al . , 2000) . To assess attachment, S. coelicolor and S. lividans were grown in 96-well flat-bottomed microtiter plates (Costar, Corning Incorporated, USA) containing 200 μl NMMP (Kieser et al . , 2000; in the absence of PEG6000 and using 50 mM glucose as a carbon source) . Prior to inoculation, spores, stored at
-20 °C in 20% glycerol, were washed and diluted in medium to a final concentration of 5 • 10s spores ml"1. 96 well flat- bottomed microtiter-plates were filled with 200 μl spore suspension per well .
TABLE 1. Plasmids Plasmid Description Reference
pGEM-T Linear plasmid used for cloning PCR fragments amplified with Promega TAQ polymerase in E. coli pBluescript KS+ pUC18 derivative for cloning in E. coli Stratagene pZero-2.1 Invitrogen pC46a pBluescript KS+ containing a 4,5 kb Sail fragment of cosmid 46 This work (Redenbachet αt, 1996) of Streptomyces coelicolor encompassing the coding sequences of rdlA and rdlB , their interspersed promoter region and flanking regions of 0,8 kb (rdlA) and 2,5 kb (rdlB)
PC46b pZero2.1 containing the Sail fragment described for pC46a This work pC46c pC46b derivative carrying a 1,4 kb Smal fragment containing a This work hygromycine resitance cassette (Zalacain . al., 1986) replacing a 0,8 kb Blpl I Seal fragment encompassing rdlA, the 5' end of the coding sequence of rdlB as well as their interspersed promoter region pC46d pC46c derivative carrying a 1.8 kb apramycin resistance cassette This work contained on a Smal fragment (Prentki and Krisch, 1984) cloned in the Xbal site of pC46c
MOLECULAR TECHNIQUES
Standard molecular techniques followed Sambrook et al . (1989) . Protoplast preparation and transformation were performed as described by Kieser et al . (2000) using alkali- denatured DNA (Oh and Chater, 1997) . The rich solid medium R2YE was used for regenerating protoplasts overlaid with the appropriate antibiotic after 18 hrs . Chromosomal DNA of S. coelicolor and S. lividans was isolated according to Verhasselt et al . , (1989) and modified by Nagy et al . (1995) . Total RNA of S. coelicolor and S. lividans was isolated using the SV Total RNA Isolation System (Promega) . DNA and RNA were blotted on Nylon filters (Boehringer, Mannheim) and
hybridised under conditions described by Church and Gilbert (1984) at 60 °C. Under these conditions rdlA and rdlB do not cross-hybridise. Radioactively labelled probes were made using the oligolabelling kit (Pharmacia, Uppsala) . ISOLATION OF THE rdlA AND rdlB GENES FROM S. coelicolor A3 (2) AND S. lividans TK23 To isolate rdl genes from S. coelicolor A3 (2) and S. lividans, a degenerate oligonucleotide
(SGCSGASAGSACSGASAGGTCCTCSAGSACGTGSGASAGSGCGCCGTC) represent- ing the N-terminal sequence of an internal peptide of RdlA of S. lividans (see Results) was radioactively labelled and hybridised to the cosmid library of S. coelicolor A3 (2) (Redenbach et al . , 1996).
CONSTRUCTION OF THE rdlAB GENE DELETION PLASMID
The rdl genes were deleted by replacing a 0.8 kb Blpl/Scal fragment of pC46b (see Table 1) containing the entire coding sequence of rdlA and 136 bp of rdlB as well as the interspersed promoter region by a 1.4 kb Smal fragment containing the hygromycin B resistance gene (Zalacaϊn et al . , 1986) . This resulted in vector pC46c. A 1.8 kb Smal fragment containing the apramycin resistance gene was cloned in the Xbal site of pC46c to select for double crossover events, resulting in plasmid pC46d.
PREPARATION OF CELL WALLS AND PROTEIN EXTRACTS Filaments of S. coelicolor A3 (2) and S. lividans were fragmented at 20,000 psi using a SLM FrenchΦ Pressure Cell Press. Cell walls were treated with 2% SDS at 100 °C as described (Wessels et al . , 1991a) (Wessels et al . , 1991b) and subsequently extracted with TFA (Wδsten et al . , 1993) . After removal of the solvent by a stream of air, extracts were taken up in SDS sample buffer (2% SDS, 20% glycerol , 0.02% bromophenolblue, 0.1 M Tris-HCl, pH 6.8) and subjected to SDS-PAGE. If necessary, adjustments of pH were done by addition of 25% ammonia. RdlA and RdlB were purified by taking up TFA extracts of SDS-treated cell walls in water without shaking. Insolubles were removed by centrifugation at 10,000 g for 15 minutes.
ELECTROPHORESIS AND WESTERN BLOTTING SDS-PAGE was done in 16% SDS-polyacrylamide gels. Prestained broad range molecular weight markers of Biorad were used. After separation, proteins were stained with 0.25% Coomassie Brilliant Blue G-250 (CBB) or blotted onto a polyvinylidenedifluoride (PVDF) membrane using semi -dry blotting. For N-terminal sequencing, a PVDF membrane was stained with CBB and a slice of the membrane containing the protein was excised. After destaining with 30% methanol, the N-terminal sequence was determined using a pulse liquid sequenator on line connected to a PTH analyser (Eurosequence, Groningen, The Netherlands) . To determine N-terminal sequences of internal peptides, the protein was eluted from the SDS-PAA gel followed by a tryptic digestion. Peptides were sequenced after separation on a C18 reversed phase HPLC column.
Antibodies against RdlA and RdlB were raised by injecting a mixture of these proteins of S. lividans eluted from a SDS- PAA gel. PVDF membranes were treated with diluted anti- RdlA/RdlB serum (1:1000) as described (Harlow and Lane, 1988) .
IMMUNOLOCALIZATION Fixation, embedding and immuno-labelling of cultures were performed as described (Wόsten et al . , 1994) with the modification that K4M was substituted for Unicryl . Polyclonal antibodies raised against a mixture of RdlA and RdlB of S. lividans were purified with an acetone powder of mycelium (Harlow et al . , 1988) of a liquid shaken culture and diluted 1000 times.
ATTACHMENT ASSAY
To quantify attachment of S. coelicolor and S. lividans 25 μl 0.5% crystal violet (Acros Organics) was added to each well and incubated for 10 minutes to stain cell material. This was followed by washing three times with 200 μl of water using a Vaccu-Pette/96 (Sigma) , removing all non- adherent cells. After drying overnight at 30 °C the adherence of cells was quantified by solubilizing the crystal violet
with 200 μl of 10% SDS (Reynolds and Fink, 2001) during 30 minutes under shaking conditions (900 rpm) . 100 μl was transferred to a new well and the OD570 was determined using a microtiter plate reader. If necessary, dilutions were made in 10% SDS. Total biomass formed was determined using Lowry with the modification that cells were treated at 100 °C for 30 minutes in 0.2% SDS/l M NaOH. BSA was used as a standard. The relative attachment was determined by comparison of the ODS70 of wildtype and disruptant strains.
RESULTS
Identification of an abundant SDS-insoluble cell wall protein specifically present in aerial structures of S . coelicolor and S . lividans Cell walls of a 5-day-old sporulating culture of
Streptomyces lividans grown on solid MS medium were treated with 2% SDS at 100 °C. After washing with water and lyophilizing, this was followed by an extraction with trifluoroacetic acid (TFA) . SDS-PAGE of the SDS-soluble fraction showed a complex pattern of polypeptides (results not shown) . Among the proteins that were insoluble in hot SDS but soluble in TFA, an abundant polypeptide, called Rdl, was observed with an apparent molecular weight of 18 kDa (Figure 1, left panel). Western analysis with antibodies raised against this protein showed the absence of it in a TFA extract of SDS-treated walls of liquid shaken cultures and in the SDS-soluble fractions of shaken and solid cultures (results not shown) . In addition, it was shown that the presence of Rdl correlated with the presence of aerial hyphae. In cell walls of 1-day-old cultures not yet forming aerial hyphae Rdl was absent (data not shown) . In contrast, the protein was abundantly present in cultures that had formed a confluent layer of aerial hyphae.
When water instead of 2% SDS was added to the TFA extract of SDS-treated cell walls of cultures forming aerial hyphae, Rdl was the main protein that dissolved (Figure 1, right panel) . In water the extract formed an SDS-insoluble complex upon shaking that could be dissociated with TFA. Similar results as obtained with S. lividans were obtained
with cultures of S. coelicolor. These data indicate that, under physiological conditions, Rdl is an insoluble cell wall protein specifically present in cultures of S. lividans and S. coelicolor forming aerial structures.
CLONING AND CHARACTERIZATION OF THE rdl GENES OF S. lividans AND S. coelicolor
N-terminal sequencing revealed that the Rdl protein of S. lividans running at the 18 kDa position was in fact a mixture of two similar proteins, called RdlA and RdlB, with slightly different N-termini (Seq. 1 starting at amino acid 29 and Seq. 2 starting at amino acid 29) . In addition, N- termini of two internal peptides were determined that resulted from a tryptic digestion of a mixture of RdlA and RdlB. A radioactive degenerated oligonucleotide based on one of the peptides was used to screen a cosmid library of S. coelicolor A3 (2) (Redenbach et al . , 1996). The oligonucleotide hybridised to the overlapping cosmids C46 and C61. The hybridising fragment of cosmid C46 was contained on a 4.5 kb Sail fragment. This fragment was cloned in pBluescript KS+ in the unique Sail site, and introduced in E. coli DH5a (deposited on 14 March, 2000 under accession number CBS 102638 with Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, The Netherlands) and partially sequenced. An ORF was identified that encodes a polypeptide of 131 aa. It starts with a putative signal sequence for secretion followed by a sequence corresponding to the determined N-terminal sequence of RdlA as found in the cell wall (mature RdlA) . The N-terminal sequences of both internal peptides were also identified in the ORF. The rdlB gene (Seq. 4) , divergently transcribed from rdlA (Seq. 3) , was identified 262 bp upstream of the start codon of rdlA . It encodes a protein very similar to that encoded by rdlA (68.7% identity, 14.5% similarity) and contains the N-terminus of mature RdlB preceded by a putative signal sequence of 28 amino acids. Sequences 3 and 4 represent the coding sequences of rdlA and rdlB including the leader or signal peptide of 28 amino acids (starting at GTG) .
The coding sequences of rdlA and rdlB hybridised to
the same unique fragments of genomic DNA of S. coelicolor and S. lividans digested with a variety of enzymes (one at a time) . For instance, a 4.5 kb Sail fragment hybridised to both rdlA and rdlB . A slightly larger genomic fragment hybridised after digestion with Blpl , while digestion with Pstl resulted in a fragment of about 8 kb (data not shown) . The almost complete genome sequence of S. coelicolor (http://www.sanger.ac.uk/Projects/S_coelicolor/) did not reveal homologous sequences of rdlA and rdlB . Using PCR and primers based on the gene sequences of rdlA and rdlB of S. coelicolor, the sequences of the homologues of S. lividans were isolated and appeared to be identical to the sequences of S. coelicolor. Digested genomic DNA of Streptomyces tendae and Streptomyces griseus hybrid- ized with a probe directed against the coding sequence of rdlA (data not shown) . In S. tendae two fragments of 4.2 and 2.8 kb hybridized while in S. griseus fragments of 3.1 , 2.2 and 1.0 kb reacted. Hybridization with rdlB showed that also homologues of this gene are present in these streptomycetes (data not shown) . rdlA AND rdlB ARE EXPRESSED IN AERIAL HYPHAE To determine the temporal expression of rdlA and rdlB total RNA was isolated from cultures of S. coelicolor and S. lividans grown in liquid YEME medium or on MS plates. After separation on a formaldehyde gel and blotting to a
Nylon membrane, RNA was hybridised with a probe representing the coding sequence of rdlA or rdlB (Figure 2) . Expression of both rdl genes correlated with the formation of aerial hyphae. rdl mRNA was not detected in 1-day-old cultures (lower panel lane 1) not yet forming aerial hyphae, nor in 3,
4, and 7 -day-old sporulating cultures (lower panel, lanes 3- 5) . However, it accumulated to high levels in 2 -day-old cultures forming aerial hyphae (lower panel, lane 2) . To verify that equal amounts of RNA were loaded in each lane of the gel, the Northern was hybridized with a probe representing 16S rRNA (upper panel) . Accumulation of rdl mRNA was not observed in liquid shaken cultures (not shown) .
RdlA AND RdlB ARE LOCALIZED AT THE OUTER SURFACE OF
AERIAL HYPHAE AND SPORES
RdlA and RdlB were localized using an antiserum raised against a mixture of RdlA and RdlB of S. lividans . In cross sections of cultures of S. lividans and S. coelicolor grown on solid MS medium immuno-gold-labelling was observed at the outer surface of cell walls of aerial hyphae (Figure 3) and spores, but not of submerged hyphae (data not shown) . The reactive layer at the outside of the wall was sometimes detached, indicating that it is a discrete layer. The antiserum did not react with cultures not forming aerial hyphae, i.e. cultures of S. lividans and S. coelicolor grown in liquid shaken medium, and 1-day-old cultures grown on solid medium.
DISRUPTION OF rdlA AND rdlB DOES NOT AFFECT FORMA¬
TION OF AERIAL HYPHAE
The rdlA and rdlB genes were deleted in Streptomyces coelicolor and Streptomyces lividans with the deletion construct pC46d (see Methods) . Gene replacement was confirmed by Southern analysis. A genomic 4.5 kb Sail fragment of the wildtype strains hybridizes to probes against rdlA and rdlB . In the mutant strains an expected 3.2 kb fragment hybridized with a probe against rdlB while no hybridization was observed, as expected, with a probe against rdlA (data not shown) . Germination, growth rates and differentiation of aerial hyphae into spores of wildtype and mutant strains were similar in different media and culture conditions (data not shown) . In addition, the characteristic rodlet layer was observed at surfaces of aerial hyphae and spores in both the wildtype and the mutant strains (a.k.a. disruptants, which lack the rdl genes) .
DISRUPTION OF THE rdl GENES AFFECTS ATTACHMENT OF
HYPHAE TO POLYSTYRENE
Since RdlA and RdlB form a discrete surface layer on aerial structures (i.e. structures formed in a hydrophobic environment in this case the air) , we tested disruptants of S. coelicolor and S. lividans for their capacity to adhere to the hydrophobic surface of standard 96 well microtiter plates of polystyrene. Strains were grown in liquid medium in the 96
well plates and attachment of hyphae was visualized with crystal violet (Figure 4 Upper part) . Attachment of the mutant strains S. coelicolor ΔAB6 and S. lividans ΔAB3 was only 50-80% throughout culturing compared to that of the wildtype strains (Figure 4, graph) . Reintroduction of rdlA in the S. coelicolor disruptant strain was sufficient to restore attachment to the hydrophobic solid (not shown) .
EXPRESSION OF Rdl PROTEINS As desired, the genes coding for the protein according to the invention may be introduced into a vector which is used to transform a host suitable for obtaining the protein in good yield and allowing for easy purification with the desired purity. It is anticipated that for hosts excreting the protein according to the present invention even culture medium may be used to coat a surface. By eliminating the protein-producing organism, the supernatant qualifies as containing at least partially purified protein.
TEFLON CAN BE MADE WETTABLE USING RdlA AND RdlB When a piece of Teflon was incubated overnight in the water-soluble fraction of the TFA extract of SDS-treated cell walls of S. lividans or S. coelicolor isolated from cultures forming aerial hyphae (and thus containing RdlA and RdlB (40 μg/ml) ; Figure 1, right lane), the Teflon became wettable showing that the protein coats the Teflon. This demonstrates the applicability of the protein for coating surfaces .
REFERENCES
1. Church, G.M., et al . (1984) Proc . Natl . Acad . Sci . USA 81: 1991-1995.
2. Harlow, E., et al . (1988) Antibodies : a Laboratory Manual Cold Spring Harbor: Cold Spring Harbor Laboratory. 3. Kieser, T., et al . (2000) Practical Streptomyces genetics Norwich, United Kingdom: The John Innes Foundation.
4. Nagy, I. et al . (1995) J. Bacteriol . 177: 676-687.
5. Oh, S.-H. et al. (1997). J. Bacteriol . 179: 122-127.
6. Prentki, P. et al . (1984) Gene 29: 303-313. 7. Redenbach, M. et al . (1996). Mol . Microbiol . 21: 77-96.
8. Reynolds, T.B. et al . Science 291: 878-881.
9. Richter, M. , et al . (1998) FEMS Microbiol . Lett . 163: 165-171.
10. Sambrook, J. et al . (1989) Molecular cloning: a labora - tory manual Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press .
11. Verhasselt, P. et al . J. (1989). FEMS Microbiol . Lett . 50: 135-140.
12. Wessels, J.G.H. et al . (1991b). Plant Cell 3: 793-799. 13. Wessels, J.G.H. et al . (1991a). J". Gen . Microbiol . 137:
2439-2445.
14. Wessels, J.G. H. et al . (1997). Adv. Microb . Physiol . 38: 1-45.
15. Wόsten, H.A.B. et al . (1994). Eur. J. Cell . Biol . 63: 122-129.
16. Wδsten, H.A.B. et al . (1993). Plant Cell 5: 1567-1574.
17. Zalacain, M. et al . (1986). Nucleic Acids Res . 14: 1565- 1581.
SEQUENCE LISTING
<110> Biomade B.V.
<120> Protein capable of self-assembly at a hydrophobic-hydrophylic interface
<130> GB44441
<140> <141>
<160> 4
<170> Patentln Ver. 2.1
<210> 1 <211> 131 <212> P T <213> Artificial Sequence
<220>
<221> SIGNAL <222> (1) .. (28)
<400> 1
Met Leu Lys Lys Ala Met Val Ala Ala Ala Ala Ala Ala Ser Val lie 1 5 10 15
Gly Met Ser Ala Ala Ala Ala Pro Gin Ala Leu Ala lie Gly Asp Asp
20 25 30
Asn Gly Pro Ala Val Ala Asn Gly Asn Gly Ala Glu Ser Ala Phe Gly 35 40 45
Asn Ser Ala Thr Lys Gly Asp Met Ser Pro Gin Leu Ser Leu Val Glu 50 55 60
Gly Thr Leu Asn Lys Pro Cys Leu Gly Val Glu Asp Val Asn Val Ala 65 70 75 80
Val lie Asn Leu Val Pro lie Gin Asp lie Asn Val Leu Ala Asp Asp 85 90 95
Leu Asn Gin Gin Cys Ala Asp Asn Ser Thr Gin Ala Lys Arg Asp Gly 100 105 110
Ala Leu Ser His Val Leu Glu Asp Leu Ser Val Leu Ser Ala Asn Gly 115 120 125
Glu Gly Arg 130
<210> 2
<211> 133 <212> PRT <213> Streptomyces coelicolor
<220>
<221> SIGNAL <222> (1) .. (28)
<400> 2 Met He Lys Lys Val Val Ala Tyr Ala Ala He Ala Ala Ser Val Met 1 5 10 15
Gly Ala Ser Ala Ala Ala Ala Pro Gin Ala Met Ala He Gly Asp Asp 20 25 30
Ser Gly Pro Val Ser Ala Asn Gly Asn Gly Ala Ser Gin Tyr Phe Gly 35 40 45
Asn Ser Met Thr Thr Gly Asn Met Ser Pro Gin Met Ala Leu He Gin 50 55 60
Gly Ser Phe Asn Lys Pro Cys He Ala Val Ser Asp He Pro Val Ser 65 70 75 80
Val He Gly Leu Val Pro He Gin Asp Leu Asn Val Leu Gly Asp Asp
85 90 95
Met Asn Gin Gin Cys Ala Glu Asn Ser Thr Gin Ala Lys Arg Asp Gly 100 105 110
Ala Leu Ala His Leu Leu Glu Asp Val Ser He Leu Ser Ser Asn Gly 115 120 125
Glu Gly Gly Lys Gly 130
<210> 3 <211> 396 <212> DNA
<213> Streptomyces coelicolor
<400> 3 gtgctcaaga aggcaatggt cgccgcggcg gctgccgctt ctgtgatcgg catgtcggct 60 gccgccgctc cccaggccct ggccatcggg gacgacaacg ggccggccgt ggccaacggc 120 aacggcgccg agtcggcgtt cggcaactcg gccaccaagg gcgacatgag cccccagctg 180 tcgctggtcg agggcacgct gaacaagccg tgcctcggtg tcgaggacgt caacgtcgcc 240 gtcatcaacc tcgtgccgat ccaggacatc aacgtcctgg cggacgacct gaaccagcag 300 tgcgcggaca actccacgca ggccaagcgg gacggcgccc tgtcgcacgt cctggaggac 360 ctgtcggtgc tgtcggcgaa cggcgagggc cgctga 396
<210> 4 <211> 402 <212> DNA
<213> Streptomyces coelicolor
<400> 4 gtgatcaaga aggtagttgc ctacgcggcg atcgccgcct ccgtcatggg tgcctccgct 60 gccgcggccc cgcaggcgat ggcgatcggc gacgacagcg ggcccgtctc cgccaacggg 120 aacggcgcct cgcagtactt cggcaactcg atgaccacgg gcaacatgag cccgcagatg 180 gcgctcatcc agggctcgtt caacaagccg tgcatcgcgg tcagcgacat cccggtcagt 240 gtcatcggtc tggtgccgat ccaggacctc aacgtcctgg gcgacgacat gaaccagcag 300 tgcgccgaga actcgacgca ggccaagcgc gacggtgcgc tggcccacct cctggaggac 360 gtctcgatcc tgtcctccaa cggcgagggc ggcaagggct ga 402
Claims
1. Protein capable of self-assembly at a hydrophobic- hydrophilic interface, characterized in that the protein is an at least partially purified protein comprising a polypeptide having at least 40% identity and at least 5% similarity with at least one polypeptide chosen from the group consisting of i) amino acids 29 - 131 of SEQ NO. 1 and ii) amino acids 29 - 133 of SEQ. NO. 2.
2. Protein according to claim 1, characterized in that the protein is derived from a filamentous bacterium.
3. Protein according to claim 2, characterized in that the filamentous bacterium is an aerial hyphae forming bacterium.
4. Protein according to claim 2 or 3 , characterized in that the filamentous bacterium is an Actinomvcete .
5. Protein according to claim 4, characterized in that the filamentous bacterium is a Streptomyces species.
6. Protein according to claim 5, characterized in that the Streptomyces species is transformed with a construct that can be isolated from an E_ coli strain which has been deposited on 14 March, 2000 under accession number CBS 102638 with the Centraalbureau voor Schimmelcultures (Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, the Netherlands) .
7. Protein according to any of the preceding claims, characterized in that the identity is at least 50%, preferab- ly at least 60% and more preferably at least 70%.
8. Protein according to any of the preceding claims, characterized in that the similarity is at least 7%, preferably at least 10% and more preferably at least 15%.
9. Method of coating a surface of an object, character- ized in that a protein according to any of the claims 1 to 8 is used.
10. Method of coating a. surface of an object according to claim 9, characterized in that the object is chosen from the group consisting of a window, a contact lens, a biosensor, a medical device, a container for performing an assay or storage, the hull of a vessel or a frame or bodywork of a car, and a solid particle wherein a solution comprising the protein according to any of the claims 1 to 8 is contacted with said object.
11. Method of stabilizing a dispersion, characterized in that a protein according to any of the claims 1 to 8 is used to stabilize the dispersed particles.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU46945/01A AU4694501A (en) | 2000-03-30 | 2001-03-30 | Protein capable of self-assembly at a hydrophobic-hydrophilic interface and usesthereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0007770.1 | 2000-03-30 | ||
GB0007770A GB0007770D0 (en) | 2000-03-30 | 2000-03-30 | Protein capable of self-assembly at a hydrophobic hydrophillic interface, method of coating a surface, method of stabilizing a dispersion, method of stabilizi |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001074864A1 true WO2001074864A1 (en) | 2001-10-11 |
Family
ID=9888793
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2001/000268 WO2001074864A1 (en) | 2000-03-30 | 2001-03-30 | Protein capable of self-assembly at a hydrophobic-hydrophilic interface and uses thereof |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU4694501A (en) |
GB (1) | GB0007770D0 (en) |
WO (1) | WO2001074864A1 (en) |
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WO2015124447A1 (en) | 2014-02-18 | 2015-08-27 | Unilever Plc | Stabilized aerated confection containing hydrophobin |
WO2016193547A1 (en) | 2015-06-02 | 2016-12-08 | Teknologian Tutkimuskeskus Vtt Oy | A method for increasing foam stability |
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AU4694501A (en) | 2001-10-15 |
GB0007770D0 (en) | 2000-05-17 |
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