CN101115768A - Novel hydrophobin fusion products, production and use thereof - Google Patents
Novel hydrophobin fusion products, production and use thereof Download PDFInfo
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- CN101115768A CN101115768A CNA200680004292XA CN200680004292A CN101115768A CN 101115768 A CN101115768 A CN 101115768A CN A200680004292X A CNA200680004292X A CN A200680004292XA CN 200680004292 A CN200680004292 A CN 200680004292A CN 101115768 A CN101115768 A CN 101115768A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A new peptide is not connected in a natural manner with an external hydrophobin and has a contact angle of at least 20[deg] during coating of the glass surface. The structural formula of a new polypeptide is: Xn-C1>-X1-50-C2>-X0-50-C3>-X1-100-C4>-X1-100-C5>-X1-50-C6>-X0-5 -C7>-X1-50-C8>-Xm, where X : amino acids; n,m : 0 - 500 and; C : cysteine. The proviso is that at least one of the peptide sequences abbreviated by Xn or Xm is a peptide sequence of at least 20 amino acids in length. The peptide is not connected in a natural manner with an external hydrophobin and has a contact angle of at least 20[deg] during coating of a glass surface. Independent claims are included for: (1) nucleic acid coding for polypeptide; and (2) procedure of polypeptide production by expressing the above nucleic acid in a host organism and isolating the obtained protein.
Description
The present invention relates to new hydrophobin fusion rotein, its preparation and purposes.
Prior art
Hydrophobin is the small protein matter of about 100 AA, and it is the distinctive protein of filamentous fungus and does not produce in other biological.In recent years, found to be called the hydrophobin-like protein of " Chaplin " in streptomyces coelicolor (Streptomycescoelicolor), it has the high surface characteristic equally.Chaplins can be in the assembling of water-aerosphere face, thereby produces amyloid fibrils (amyloid-like fibrils) (people such as Classen, 2003 Genes Dev 1714-1726; People such as Elliot 2003, Genes Dev.17,1727-1740).
Hydrophobin is distributed in the surface of various fungi structure example such as aerial hyphae, spore and sporophore with the water-insoluble form.The gene isolation of hydrophobin is from Ascomycetes (ascomycetes), deuteromycetes (deuteromycetes) and Basidiomycetes (basidiomycetes).Some fungies comprise more than a kind of hydrophobin genes, for example Split-gill (Schizophyllum commune), Coprinus cinereus (Coprinus cinereus), Aspergillus nidulans (Aspergillus nidulans).Clearly, the different steps of different hydrophobin participation fungi developments.Infer that described hydrophobin is responsible for different functions (people such as van Wetter, 2000, Mol.Microbiol., 36,201-210; People such as Kershaw 1998, Fungal Genet.Biol, 1998,23,18-33).
The biological function of described hydrophobin except the surface tension that reduces water producing the aerial hyphae, it also comprises hydrophobization (people 1999 such as W sten, Curr.Biol., 19, the 1985-88 of spore; People such as Bell 1992, Genes Dev., 6,2382-2394).In addition, hydrophobin is used for component (people 1999 such as Lugones, Mycol.Res., 103, the 635-640 of the gas passage (gas channel) of the sporophore of liner lichens and the system by fungal pathogens plant identification surface; Hamer ﹠amp; Talbot 1998, Curr.Opinion Microbiol., and the 1st volume, 693-697).
Complementation test has proved and can functionally replace hydrophobin to a certain extent in single kind.
Use conventional protein-chemical purification and separation method to prepare previously disclosed hydrophobin with output and purity only with appropriateness.Provide the effort of more substantial hydrophobin also not succeed so far by genetic method.
Goal of the invention
The purpose of this invention is to provide new hydrophobin and its production method, described method makes it possible to produce hydrophobin economically and use it for the multiple technologies field.
Invention is described
The present invention relates to the polypeptide of universal architecture formula (I)
X
n-C
1-X
1-50-C
2-X
0-5-C
3-X
1-100-C
4-X
1-100-C
5-X
1-50-C
6-X
0-5-C
7-X
1-50-C
8-X
m (I)
Wherein X can be any (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile, Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, the Gly) in the amino acid of 20 kinds of natural generations, the amino acid whose number of exponential representation on the X, index n and m are the numbers between the number, preferred 15 and 300 between 0 and 500, and condition is to be abbreviated as X
nOr X
mPeptide sequence at least one be that length is at least 20 amino acid, the natural peptide sequence that does not link to each other with hydrophobin, this polypeptide contact angle after bag is by glass surface changes at least 20 °.
By C
1To C
8The halfcystine of expression exists with the reduction form in protein of the present invention or forms disulfide linkage each other.Be preferably formed intramolecularly C-C key especially, particularly have at least 1, preferred 2, preferred especially 3 and very preferably 4 be selected from: C
1And C
2, C
3And C
4, C
5And C
6, C
7And C
8The C-C key of intramolecular disulfide bond.
If halfcystine also is used for the position that X represents, the numbering of each halfcystine position can correspondingly change in the general formula so.
Particularly advantageous polypeptide is the those polypeptides of general formula (II):
X
n-C
1-X
3-25-C
2-X
0-2-C
3-X
5-50-C
4-X
2-35-C
5-X
2-15-C
6-X
0-2-C
7-X
3-35-C
8-X
m (II)
Wherein X can be any (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile, Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, the Gly) in the amino acid of 20 kinds of natural generations, the amino acid whose number of exponential representation on the X, index n and m are the numbers between 2 and 300, and condition is to be abbreviated as X
nOr X
mPeptide sequence at least one peptide sequence be that length is at least 35 amino acid, the natural peptide sequence that does not link to each other with hydrophobin, this polypeptide change at least 20 ° of contact angles after bag is by glass surface.
Very advantageously be the those polypeptides of general formula (III):
X
n-C
1-X
5-9-C
2-C
3-X
11-39-C
4-X
2-23-C
5-X
5-9-C
6-C
7-X
6-18-C
8-X
m (III)
Wherein X can be any (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile, Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, the Gly) in the amino acid of 20 kinds of natural generations, the amino acid whose number of exponential representation on the X, index n and m are the numbers between 0 and 200, and condition is to be abbreviated as X
nOr X
mPeptide sequence at least one peptide sequence be the peptide sequence that length is at least 40 amino acid, does not link to each other natively with hydrophobin, this polypeptide changes at least 20 ° of contact angles after bag is by glass surface.
Embodiment preferred of the present invention is to have universal architecture formula (I), (II) or polypeptide (III), this structural formula comprises the hydrophobin of at least one kind I, preferably at least one dewA, rodA, hypA, hypB, sc3, basf1, basf2 hydrophobin or its part or derivative.The constitutional features of described hydrophobin is present in the sequence of listing below.May be with a plurality of, preferred 2 or 3, hydrophobin identical or different on the structure interconnects, and is connected to corresponding suitable peptide sequence, and described peptide sequence does not link to each other with hydrophobin is natural.
Particularly preferred embodiment of the present invention is new protein and the nucleic acid sequence encoding thereof with the peptide sequence shown in the SEQ ID NO:20,22,24, particularly as SEQ ID NO:19, and the sequence of 21,23 definition.Particularly preferred embodiment also is such protein, this protein starts from the peptide sequence shown in the SEQ ID NO:22,22 or 24, by at least 1,10 of as many as, preferred 5 amino acid, preferred especially all amino acid whose replacements of amino acid whose 5%, insertion or disappearance produce and still have at least 50% biological characteristics of described initiation protein.Proteinic biological characteristics herein is meant the change as the contact angle described in the embodiment 10.
Protein of the present invention is being abbreviated as X
nOr X
mThe position at least one on have and comprise at least 20, preferably at least 35, especially preferably at least 50 and especially preferred at least 100 amino acid whose peptide sequences (being also referred to as fusion partner hereinafter), it is natural not to link to each other with hydrophobin.This shows protein of the present invention by hydrophobin part with merge and state the fact that the companion partly forms, and wherein said fusion is stated companion's part and do not taken place together with this form at occurring in nature.
Described fusion partner part can be selected from a plurality of protein.Also may be with a plurality of fusion partners at for example N-terminal (X of hydrophobin part
n) and C-terminal (X
m) be connected to a hydrophobin part.Yet, also may for example two fusion partners be connected to the proteinic single position (X of the present invention
nOr X
m).
Particularly preferred fusion partner is such peptide sequence, be that these peptide sequences can make protein bag of the present invention by glass surface and make the processing of the anti-washing agent of handling through this protein of glass surface, as the processing of describing in detail at experimental section (embodiment 10) (for example 1%SDS/80 ℃/10 minutes).
Specially suitable fusion partner is the polypeptide of natural generation in intestinal bacteria (E.coli) or Bacillus subtilus (Bacillus subtilis) particularly in microorganism.The example of these fusion partners is sequences y aad (SEQ ID NO:15 and 16), yaae (SEQ ID NO:17 and 18) and Trx matter.The part that only comprises described sequence, preferred 70-99%, the fragment or the derivative of the described sequence of preferred especially 80-98%, or wherein compare the sequence that single amino acids or nucleic acid changed with described sequence also be very useful.For example, can be with extra amino acid, especially two extra amino acid are preferably the C-terminal that amino acid Arg, Ser are connected to yaad and yaae sequence.Compare with the sequence of natural generation, be inserted in the additional amino acid in the yaae sequence, for example the 2nd amino acids (Gly) among the SEQ ID NO:17 and 18 also may be preferred.
In addition, also may insert two extra amino acid in the junction of two fusion partners, this is the new result who produces the recognition site of restriction enzyme or make the recognition site inactivation of restriction enzyme on nucleic acid level.
Also may for example modify proteinic peptide sequence of the present invention by glycosylation, acetylize or by chemically crosslinked by glutaraldehyde cross-linking.
A proteinic characteristic of the present invention is when with described protein bag during by the surface, and described surface properties changes.Can be by measuring the contact angle of water droplet with protein bag of the present invention before or after by the surface and determining that the difference of two measurements tests the change of determining described surface properties.
Illustrated in the experimental section in embodiment 10 that being used to measure contact angle tests condition really conscientiously.Under these conditions, protein of the present invention has the characteristic that increases contact angle at least 20 degree, preferred 25 degree, preferred especially 30 degree.
Guard the polarity of the hydrophobin of disclosed hydrophobin part and the position of nonpolar amino acid before, thereby produce the feature hydrophobicity profile.Disclosed hydrophobin was divided into two classes before bio-physical property and hydrophobic difference made it, and I class and II class (people 1994 such as Wessels, Ann.Rev.Phytopathol., 32,413-437).
The assembling film of I class hydrophobin is highly insoluble (even insoluble for 1% SDS under the temperature that improves), and it uses spissated trifluoroacetic acid (TFA) or formic acid to dissociate once more only.On the contrary, the assembling form of II class hydrophobin is unstable.Even the SDS of the ethanol of available 60% intensity or 1% (at room temperature) is again with its dissolving.
Aminoacid sequence relatively show halfcystine C in the II class hydrophobin
3And C
4Between the length in zone be significantly shorter than its length in I class hydrophobin.
In addition, II class hydrophobin has more charged amino acid than I class.
The invention still further relates to production method of protein of the present invention.Can for example chemically produce these polypeptide by known peptide synthetic method according to the solid-phase synthesis of Merrifield.
Yet genetic method is useful especially, in described genetic method, combination two nucleotide sequences dna sequence dna particularly of fusion partner and hydrophobin part of encoding respectively by this way is even the genetic expression of the nucleotide sequence that makes up in host living beings produces the proteinic mode of wanting.
Appropriate host biology (producer's biology) can be prokaryotic organism (comprising archeobacteria (Archaea)) or eukaryote herein, particularly bacterium comprises salt bacillus (halobacteria) and methane coccus (methanococci), fungi, insect cell, vegetable cell and mammalian cell, preferred especially intestinal bacteria, Bacillus subtilus, Bacillus megatherium (Bacillus megaterium), aspergillus oryzae (Aspergillus oryzea), Aspergillus nidulans, aspergillus niger (Aspergillus niger), pichia pastoris phaff (Pichia pastoris), pseudomonas species (Pseudomonas spec.), Lactobacillen, multiple-shaped nuohan inferior yeast (Hansenula polymorpha), Trichodermareesei (Trichoderma reesei), SF9 (or relevant cell) and other biological.
The invention still further relates to expression construct, and the carrier that comprises at least one described expression construct, described expression construct is included in the nucleotide sequence of coding polypeptide of the present invention under the Gene Handling of regulation and control nucleotide sequence.
Promotor 5 ' the upstream and the terminator sequence 3 ' downstream that comprise the specific coding sequence, if suitable, this class construct of the present invention that also is included in the conventional controlling element that effectively is connected to described encoding sequence under each situation is preferred.
" effectively connect " if be meant series arrangement promotor, encoding sequence, terminator and suitable regulating and controlling sequence in addition by this way, thereby make each element of controlling element realize its function according to the purposes relevant of its hope with expressing described encoding sequence.
The example of the sequence that can effectively connect is target sequence and enhanser, polyadenylation signal etc.Other controlling elements comprise selective marker, amplified signal, replication orgin etc.The case description of suitable regulating and controlling sequence is in Goeddel, Gene Expression Technology:Methods inEnzymology 185, Academic Press, San Diego, CA (1990).
Except these regulating and controlling sequences, the natural regulation and control of these sequences can still be present in the upstream of practical structures gene, and if suitable, it has carried out genetic modification, has closed natural regulation and control like this, thereby expression of gene is increased.
Preferred nucleic acid construct also advantageously comprises the enhancer sequence that one or more have been mentioned, and described enhancer sequence functionally is connected to promotor and the expression of described nucleotide sequence is increased.Other favourable sequences for example other controlling elements or terminator also can be inserted into 3 ' end of described dna sequence dna.
Nucleic acid of the present invention also can be present in the construct with one or more copies.If suitable, construct still can comprise other marks for example antibiotics resistance or with auxotroph complementary gene, to select the described body of building of structure.
The example that helps the regulating and controlling sequence of the inventive method is present in promotor for example in cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq-T7, T5, T3, gal, trc, ara, rhaP (rhaPBAD) SP6, λ-PR or the λ-P promotor, and described promotor is advantageously used in the gram-negative bacteria.Other examples of favourable regulating and controlling sequence are present among gram-positive promotor amy and the SP02, are present among yeast or fungal promoters ADC1, MF α, AC, P-60, CYC1, GAPDH, TEF, rp28, the ADH.
Also may use artificial promotor to regulate and control.
In order in host living beings, to express, advantageously nucleic acid construct is for example inserted carrier, plasmid or phage, described carrier make gene can be in the host optimum expression.Except plasmid and phage, carrier also refers to any other carrier well known by persons skilled in the art, promptly for example, virus is SV40, CMV, baculovirus and adenovirus, transposon, IS element, phasmid, clay and linearity or annular DNA for example, also has the Agrobacterium system.
These carriers can independently duplicate or pass through chromosome duplication in host living beings.These carriers have been formed another embodiment of the invention.The example of suitable plasmid is the pLG338 in the intestinal bacteria, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III " 3-B1; tgt11 or pBdCl; the pIJ101pIJ364 in the streptomyces (Streptomyces); pIJ702 or pIJ361; the pUB110 in the Bacillus (Bacillus); pC194 or pBD214, A77 or pAJ667 in the rod bacillus (Corynebacterium), pALS1 in the fungi, pIL2 or pBB116,2 α in the yeast, pAG-1, YEp6, pLGV23 in YEp13 or pEMBLYe23 or the plant, pGHIac+, pBIN19, pAK2004 or pDH51.Described plasmid is that the minority of possible plasmid is selected.Other plasmids be to those skilled in the art know and can in for example books Cloning Vectors (people such as Pouwels P.H. writes, Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018), find.
In order to express the gene of other existence, advantageously, nucleic acid construct also comprises 3 ' extraly-and/or 5 '-terminal regulating and controlling sequence express to increase, depend on the host living beings of selection and the described regulating and controlling sequence of gene Selection to carry out optimum expression.
These regulating and controlling sequences make it possible to specifically expressing gene and protein.Depend on host living beings, this can be meant that for example, described gene is only inducing the back to express or overexpression, or expresses immediately and/or overexpression.
In this relation,, the described regulating and controlling sequence or the factor increase its expression thereby preferably can having useful effect to the genetic expression of inductive gene.Therefore, can transcribe signal for example promotor and/or enhanser by force, on transcriptional level, advantageously strengthen controlling element by using.Yet, in addition, also may increase translation by the stability that for example improves mRNA.
In another embodiment of carrier, the carrier that mode that also can be by allos or homologous recombination advantageously will comprise nucleic acid construct of the present invention or nucleic acid of the present invention with the form of linear DNA imports microorganism and is integrated in the genome of host living beings.Described linear DNA can by linearizing carrier for example plasmid form or only form by nucleic acid construct of the present invention or nucleic acid.
In order in biology, to obtain the optimum expression of heterologous gene, advantageously select to modify described nucleotide sequence according to specific cryptosystem that uses in the described biology.Can determine that easily codon selects based on the Computer Analysis of other knowns of purpose biology.
By being merged, suitable promotor and suitable coding nucleotide sequence and terminator signal or polyadenylation signal prepare expression cassette of the present invention.For this purpose, use reorganization and the clone technology be familiar with, at for example T.Maniatis, E.F.Fritsch and J.Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY (1989), with at T.J.Silhavy, M.L.Berman and L.W.Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY (1984) and at Ausubel, people such as F.M., these technology of describing among the Current Protocolsin Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987)..
In order in the appropriate host biology, to express, advantageously described recombinant nucleic acid construct or gene construct are inserted the host specificity carrier, described carrier make gene can be in the host optimum expression.Carrier is known to those skilled in the art, and can find in for example " Cloning Vectors " (people such as Pouwels P.H. writes Elsevier, Amsterdam-New York-Oxford, 1985).
Carrier of the present invention can be used for preparing the recombinant microorganism that transforms with for example at least a carrier of the present invention and can be used for producing polypeptide of the present invention.Advantageously, above-mentioned recombinant precursor of the present invention is imported the appropriate host system and in described system, express.Preferred herein use clone commonly used well known by persons skilled in the art and for example co-precipitation of transfection method, protoplastis fusion, electroporation, reverse transcription transfection etc. are to express described nucleic acid in specific expression system.Suitable system description is in for example Current Protocols in Molecular Biology, people such as F.Ausubel write, WileyInterscience, New York 1997, or people such as Sambrook, Molecular Cloning:ALaboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY is in 1989.
Microorganism that also may homologous recombination produced according to the present invention.For this purpose, preparation comprises at least one segmental carrier of gene of the present invention or encoding sequence, in the fragment of described gene,, imported at least one amino acid whose disappearance, interpolation or replaced and for example functionally destroyed sequence of the present invention (knockout carrier) to modify if suitable.The sequence of described importing also can be for example to derive from the homologue of related microorganisms or from Mammals, yeast or insect source.Selectively be designed for homologous recombination carrier so that native gene undergo mutation, or during homologous recombination, modify in some other modes, but described native gene is encoding function protein (for example, can modify upstream regulatory sequence in the mode of the expression that changes endogenous protein) still.The modified part of gene of the present invention is present in the homologous recombination vector.The structure that is used for the suitable carriers of homologous recombination is described in for example Thomas, and K.R. and Capecchi are among M.R. (1987) the Cell 51:503.
Any protokaryon or eukaryote are suitable as the recombinant host biology of nucleic acid of the present invention in principle or are used for nucleic acid construct.Advantageously the host living beings of Shi Yonging is for example bacterium, fungi or a yeast of microorganism.Advantageously use gram-positive or gram-negative bacteria, be preferably enterobacteriaceae (Enterobacteriaceae), pseudomonadaceae (Pseudomonadaceae), Rhizobiaceae (Rhizobiaceae), the bacterium of Streptomycetaceae (Streptomycetaceae) or Nocardiaceae (Nocardiaceae), be preferably escherichia especially, Rhodopseudomonas (Pseudomonas), streptomyces (Streptomyces), Nocardia (Nocardia), bulkholderia cepasea (Burkholderia), Salmonellas (SalmoneHa), the bacterium of Agrobacterium (Agrobacterium) or Rhod (Rhodococcus).
Based on host living beings, be used for the biology of the inventive method with method known to those skilled in the art growth or cultivation.Usually under the temperature between 0 and 100 ℃, between preferred 10 and 60 ℃ with in the microorganism culturing liquid medium within, it feeds oxygen simultaneously, described substratum comprises the carbon source that usually exists with the sucrose form, usually with organic nitrogen source yeast extract for example, or salt for example iron, manganese, magnesium salts of the nitrogenous source that exists of ammonium sulfate form, trace element for example, if with suitable, VITAMIN.The pH of nutritive medium can keep herein or can not remain on the fixed value, is promptly regulated and control in process of growth.Can cultivate by batchwise, semi-batch fermentation method or continuous fermentation method.Can add nutrition or semicontinuous subsequently or nutrition is provided continuously at the very start in when beginning fermentation.Can use the method for describing among the embodiment from the bioseparation enzyme, or described enzyme is used for reaction as crude extract.
The invention still further relates to reorganization and produce polypeptide of the present invention or its method functional, biological active fragment, this method comprises cultivates the microorganism that produces polypeptide, if suitable, comprise and expresses described polypeptide and it is separated from culture.If expectation also can be produced described polypeptide by this method on technical scale.Can cultivate and the fermentation recombinant microorganism by known method.For example, can be in TB substratum or LB substratum, with 20 to 40 ℃, pH6 to 9 breeds bacterium.At for example T.Maniatis, E.F.Fritsch and J.Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY has described suitable culture condition in detail in (1989).
If polypeptide is not secreted to substratum, then smudge cells also passes through currently known methods separated product from lysate of isolated protein.Optional by high frequency ultrasound, by high pressure (for example in French press), by hypotonic (osmolysis), by washing agent, lyase or organic solvent effect, by using homogenizer or passing through the combined crushing cell of listed several method.
Can be by known method, chromatography is molecular sieve chromatography (gel-filtration) for example, for example Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and by other for example ultrafiltration, crystallizations of method commonly used, saltout, dialysis and the described polypeptide of native gel electrophoresis purifying.At for example Cooper, F.G., Biochemische Arbeitsmethoden[original header: The tools ofbiochemistry], Verlag Water de Gruyter, Berlin, New York or at Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg has described suitable method among the Berlin.
For separating recombinant proteins matter, can advantageously use for example carrier system or oligonucleotide, described carrier system or oligonucleotide extend cDNA by specific nucleotide sequence, thus coding for example helps the polypeptide or the fusion rotein of the change of purifying.The example of the modification that these are suitable is as anchor " mark ", for example be called the modification of six Histidine anchors or can (be described in for example Harlow as the epi-position of antigen recognition by antibody, E. and Lane, D., 1988, among Antibodies:A Laboratory Manual.ColdSpring Harbor (N.Y.) Press).Other suitable marks are for example HA, calmodulin matter-BD, GST, MBD, chitin-BD, streptavidin-BD-Avi mark, Flag mark, T7 etc.These anchors can be used for described protein is connected with solid support, for example with can for example be imported chromatography column, or the polymeric matrix that imports microtiter plate or any other upholder connects.Also can obtain corresponding purification scheme from commercial affinity labelling provider.
Many bags by the fungi surface of hydrophobin (spore, sporophore, mycelium) but show microscopic inspection, be called the feature structure of " bar-shaped corpusculum (rodlet) ".Also can on the water-wetted surface (for example, glass, mica etc.) of hydrophobin bag quilt, detect similar bar-shaped corpusculum with about 10nm thickness (people such as W sten, 1993, Plant Cell, 5,1567-1574).
Be used for the special character (for example anti-washing agent is the SDS solution of 1% intensity for example) of pan coating owing to hydrophobin, these protein have the great potential that is used for many industrial application.Multiple patent documentation is mentioned the example of these application, herein with regard to the purposes of the hydrophobin example with reference to these application.
Numbering priority application people
| WO 03/10331 | 07.23.2001 | Nanosystems B.V. application |
| WO 04/00880 | 06.21.2002 | Nanosystems B.V. application |
| WO 03/84508 | 04.04.2002 | Nanosystems B.V. application |
| WO 00/40968 | 01.05.1999 | Unilever N.V.(Hindustan Lever Ltd.) |
| EP- |
02.04.2000 | Nanosystems B.V. Stichting voor de Technische Wetenschappen application |
| EP-B 1257 571 | 02.04.2000 | Nanosystems B.V. application |
| WO 01/57066 | 02.04.2000 | Nanosystems B.V. application |
| WO 03/10331 | 07.23.2001 | Nanosystems B.V. application |
| WO 03/53383 | 12.14.2001 | L’Oreal |
Owing to lack the effective ways of production and purifying, the hydrophobin particularly industrial application of I class hydrophobin is succeedd so far.The method that starts from natural origin (spore, fungi, mycelium etc.) of Miao Shuing only produces the material (for example WO 96/41882) of the amount of mg level before.
Method by recombinant production in multiple producer's biology is proved to be complicated especially equally and makes us very dissatisfied.Hydrophobin of the present invention is with its fusion form (promptly with the fusion partner part) and all have the characteristic of the hydrophobin of expectation with its unpack format.Therefore protein of the present invention directly may be used with the form of fusion rotein, and the form with " pure " hydrophobin is used in cutting and after removing fusion partner.
If be intended to remove fusion partner, recommend potential cleavage site (the specific recognition site of protein enzyme) is introduced fusion rotein between hydrophobin part and fusion partner part.Specially suitable cutting position is those peptide sequences that do not take place in any other position of hydrophobin part and fusion partner part, can use the information biology instrument easily to determine this sequence.Useful especially for example is, the fracture that BrCN fracture on the methionine(Met) or the protein enzyme that is undertaken by factor Xa, enteropeptidase, zymoplasm, TEV (etch virus of tobacco poisonous protein enzyme) cutting mediate.
The embodiment part
Yaad-His
6/ yaaE-His
6Clone's preparation work
Carry out the polymerase chain reaction by means of oligonucleotide Hal570 and Hal571 (Hal 572/Hal 573).The template DNA that uses is the genomic dna from the bacterium Bacillus subtilus.The PCR fragment that obtains comprises the encoding sequence of Bacillus subtilus yaaD/yaaE gene and is positioned at terminal NcoI or the restricted cleavage site of BglII.Purifying is also used restriction endonuclease NcoI and BglI cutting PCR fragment.As inserting fragment and being cloned into Qiagen pQE60 carrier, used restriction endonuclease NcoI and BglII this dna fragmentation with described carrier linearizing before.With the carrier that this mode obtains, pQE60YAAD#2/pQE60YaaE#5 can be used for expressing respectively by YAAD::HIS
6And YAAE::HIS
6The protein of forming.
Hal570：gcgcgcccatggctcaaacaggtactga
Hal571：gcagatctccagccgcgttcttgcatac
Hal572：ggccatgggattaacaataggtgtactagg
Hal573：gcagatcttacaagtgccttttgcttatattcc
Embodiment 2
Yaad-hydrophobin DewA-His
6The clone
Carry out the polymerase chain reaction by means of oligonucleotide KaM 416 and KaM 417.The template DNA that uses is the genomic dna from the mould Aspergillus nidulans.The PCR fragment that obtains comprises the encoding sequence of hydrophobin genes dewA and the cleavage site of N-terminal factor Xa protein enzyme.Purifying and and usefulness restriction endonuclease BamHI cutting PCR fragment.As inserting fragment and being cloned into the pQE60YAAD#2 carrier, used restriction endonuclease BglII this dna fragmentation with described carrier linearizing before.
The carrier that is obtained, #508 can be used for expressing by YAAD::Xa::dewA::HIS
6The fusion rotein of forming.
KaM416：GCAGCCCATCAGGGATCCCTCAGCCTTGGTACCAGCGC
KaM417：CCCGTAGCTAGTGGATCCATTGAAGGCCGCAT-
GAAGTTCTCCGTCTCCGC
Embodiment 3
Yaad-hydrophobin RodA-His
6The clone
Use oligonucleotide KaM 434 and KaM 435 that plasmid #513 is cloned into plasmid #508 similarly.
KaM434：GCTAAGCGGATCCATTGAAGGCCGCATGAAGTTCTCCATTGCTGC
KaM435：CCAATGGGGATCCGAGGATGGAGCCAAGGG
Embodiment 4
Yaad-hydrophobin BASF1-His
6The clone
Use oligonucleotide KaM 417 and KaM 418 that plasmid #507 is cloned into plasmid #508 similarly.
Used template DNA is the dna sequence dna of synthetic, hydrophobin BASF1 (referring to annex).
KaM417：CCCGTAGCTAGTGGATCCATTGAAGGCCGCAT-
GAAGTTCTCCGTCTCCGC
KaM418：CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG
Embodiment 5
Yaad-hydrophobin BASF2-His
6The clone
Use oligonucleotide KaM 417 and KaM 418 that plasmid #506 is cloned into plasmid #508 similarly.
Used template DNA is the dna sequence dna of synthetic, hydrophobin BASF2 (referring to annex).
KaM417：CCCGTAGCTAGTGGATCCATTGAAGGCCGCAT-
GAAGTTCTCCGTCTCCGC
KaM418：CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG
Embodiment 6
Yaad-hydrophobin SC3-His
6The clone
Use oligonucleotide KaM464 and KaM465 that plasmid #526 is cloned into plasmid #508 similarly.
Used template DNA is the cDNA (referring to annex) of Split-gill.
KaM464：CGTTAAGGATCCGAGGATGTTGATGGGGGTGC
KaM465：GCTAACAGATCTATGTTCGCCCGTCTCCCCGTCGT
Embodiment 7
The recombination bacillus coli strain is yaad-hydrophobin DewA-His
6Fermentation
In 15ml Greiner pipe, use and express yaad-hydrophobin DewA-His
6E. coli strains system inoculation 3ml LB liquid nutrient medium.Culture with 37 ℃, was cultivated 8 hours for 200 rev/mins on vibrator.Under each situation, be used in 2 the buffered Aoron mayer flasks that the 1l of 250ml LB substratum (+100 μ g/ml penbritin) is housed of pre-culture inoculation of 1ml under each situation, and, on vibrator, cultivated 9 hours for 180 rev/mins with 37 ℃.
In the fermentor tank of 20l with the pre-culture of 0.5l (relative H
2The OD that O measures
600nm1: 10) inoculation 13.5l LB substratum (+100 μ g/ml penicillin G).At OD
60nmFor adding the 100mM IPTG of 140ml at about 3.5 o'clock.After 3 hours, fermentor tank is cooled to 10 ℃, by the centrifugal fermented liquid of removing.Cell precipitation is used to be further purified.
Embodiment 8
The recombinate purifying of hydrophobic fusion rotein
(purifying) with hydrophobin fusion rotein of C-terminal His6 mark
With 100g cell precipitation (100-500mg hydrophobin) and 50mM sodium phosphate buffer, pH7.5 mixes the cumulative volume that forms 200ml, the described cell of resuspension then.With Ultraturrax T25 type (Janke and Kunkel; IKA-Labortechnik) handled suspended substance 10 minutes, then with benzonase (Merck, Darmstadt, Germany of 500 units; Catalog number (Cat.No.) 1.01697.0001) at room temperature incubation 1 hour with degraded nucleic acid.Before cytoclasis, use glass syringe (cartridge) (P1) to filter.Two homogenizers of operation are to carry out cytoclasis and to shear remaining genomic dna (M-110EH microfluidizer under 1500 crust; Microfluidics Corp.).Centrifugal (SorvallRC-5B, the GSA rotor, the 250ml centrifugal bottle, 60 minutes, 4 ℃, 12000 rev/mins, 23000g) homogenate placed supernatant on ice after centrifugal and at the 100ml sodium phosphate buffer, the described precipitation of resuspension among the pH 7.5.Repeated centrifugation and resuspension three times, sodium phosphate buffer comprises 1% SDS during repeating for the third time.Behind resuspension, stirred the mixture 1 hour and carry out last centrifugal (Sorvall RC-5B, the GSA rotor, the 250ml centrifugal bottle, 60 minutes, 4 ℃, 12000 rev/mins, 23000g).SDS PAGE analysis revealed is present in (Fig. 1) in the supernatant liquor at last centrifugal back hydrophobin.This experiment shows that hydrophobin may be present in the form of inclusion body in the corresponding Bacillus coli cells.The supernatant that 50ml is comprised hydrophobin is used for the efficient 17-5268-02 post of 50ml nickel-sepharose (Amersham), and this post has been used 50mM Tris-Cl damping fluid, and pH 8.0 has carried out balance.With 50mM Tris-Cl damping fluid, pH 8.0 washing pillars are then with the 50mM Tris-Cl damping fluid that comprises the 200mM imidazoles, pH 8.0 wash-out hydrophobins.With 50mM Tris-Cl damping fluid, the described solution of pH 8.0 dialysis is removed imidazoles.
Fig. 1 has described the purifying of hydrophobin of the present invention:
Swimming lane 1: the solution that is used for nickel-sepharose post (dilution in 1: 10)
Swimming lane 2: merchantable thing=washing step eluate
Swimming lane 3-5: the OD280 peak of washing fraction
Hydrophobin of the present invention among Fig. 1 has the molecular weight of about 53kD.Some littler bands are represented the degradation production of described hydrophobin.
Embodiment 9
Bag quilt/evaluation with surface of hydrophobin
Preferably on respectively as the glass of hydrophilic and hydrophobic surface model and teflon resin, estimate the bag of hydrophobin or hydrophobin fusion rotein by character.
The bag of standard is tested
Glass:
The concentration of-hydrophobin: 1-100 μ g/mL
-using the 50mM sodium-acetate, pH 4 ,+0.1%Tween 20 incubation (80 ℃ of the temperature) sheet glass that spends the night
Behind-Bao the quilt, in distilled water, wash
-afterwards, use 1%SDS incubation 10 minutes down at 80 ℃
-in distilled water, wash.
Teflon resin:
-concentration: 1-100 μ g/mL
-using 10mM Tris, pH 8 ,+0.1%Tween 20 incubation (80 ℃ of temperature) the teflon resin plate that spends the night
Behind the bag quilt, in distilled water, wash
-use 0.1%Tween 20 incubations 10 minutes down at 80 ℃
-in distilled water, wash
-afterwards, incubation is 10 minutes in 80 ℃ of following usefulness 1%SDS
-in distilled water, wash.
Contact angle (showing with kilsyth basalt) at air drying sample and definite 5 μ l water droplets obtains for example following value:
Experiment (the contrast of carrying out with the yaad-DewA fusion rotein of embodiment 8: no protein; Yaad-dewA-his
6: the fusion partner of 100 μ g/ml purifying):
| At 1%SDS, after 80 ℃ | ||
| Teflon resin | Glass | |
| Contrast | 96.8 | 30 |
| yaad | 97.4 | 38.7 |
| 100μg/ml | 77.7 | 76.8 |
| 50μg/ml | 85.9 | 77.9 |
| 10μg/ml | 83.5 | 74.5 |
| 5μg/ml | 104 | 70.3 |
| 1μg/ml | 104.9 | 73 |
Embodiment 10
Bag quilt/evaluation with surface of hydrophobin
Glass (window glass, S ü ddeutsche Glas, Mannheim, Germany):
The concentration of-hydrophobin: 100 μ g/mL
-using the 50mM sodium-acetate, pH 4 ,+0.1%Tween 20 incubation (80 ℃ of the temperature) sheet glass that spends the night
Behind-Bao the quilt, in distilled water, wash
-afterwards, incubation is 10 minutes in 80 ℃ of 1%SDS that use down in the distilled water
-in distilled water, wash.
Contact angle (showing) at air drying sample and definite 5 μ l water droplets with kilsyth basalt.
At Dataphysics Contact Angle System OCA 15+, measure contact angle on software SCA 20.2.0. (in November, the 2002) device.Measure according to manufacturers instruction.
Untreated glass produces 30 ± 5 ° contact angle; Use the functional hydrophobin (yaad-dewA-his of embodiment 8
6) glass of bag quilt produces 75 ± 5 ° ° contact angle.
The sequence title is distributed to DNA and peptide sequence in the sequence list
| DewADNA and peptide sequence | SEQ ID NO：1 |
| The dewA peptide sequence | SEQ ID NO：2 |
| RodADNA and peptide sequence | SEQ ID NO：3 |
| The rodA peptide sequence | SEQ ID NO：4 |
| HypA DNA and peptide sequence | SEQ ID NO：5 |
| The hypA peptide sequence | SEQ ID NO：6 |
| HypB DNA and peptide sequence | SEQ ID NO：7 |
| The hypB peptide sequence | SEQ ID NO：8 |
| Sc3 DNA and peptide sequence | SEQ ID NO：9 |
| The sc3 peptide sequence | SEQ ID NO：10 |
| Basf1 DNA and peptide sequence | SEQ ID NO：11 |
| The basf1 peptide sequence | SEQ ID NO：12 |
| Basf2 DNA and peptide sequence | SEQ ID NO：13 |
| The basf2 peptide sequence | SEQ ID NO：14 |
| Yaad DNA and peptide sequence | SEQ ID NO：15 |
| The yaad peptide sequence | SEQ ID NO：16 |
| Yaae DNA and peptide sequence | SEQ ID NO：17 |
| The yaae peptide sequence | SEQ ID NO：18 |
| Yaad-Xa-dewA-his DNA and peptide sequence | SEQ ID NO：19 |
| The yaad-Xa-dewA-his peptide sequence | SEQ ID NO：20 |
| Yaad-Xa-rodA-his DNA and peptide sequence | SEQ ID NO：21 |
| The yaad-Xa-rodA-his peptide sequence | SEQ ID NO：22 |
| Yaad-Xa-basf1-his DNA and peptide sequence | SEQ ID NO：23 |
| The yaad-Xa-basf1-his peptide sequence | SEQ ID NO：24 |
Claims (11)
1. the polypeptide of universal architecture formula (I)
X
n-C
1-X
1-50-C
2-X
0-5-C
3-X
1-100-C
4-X
1-100-C
5-X
1-50-C
6-X
0-5-C
7-X
1-50-C
8-X
m (I)
Wherein X is any in the amino acid of 20 kinds of natural generations,
N and m are the numbers between 0 and 500,
C is a halfcystine,
Condition is to be abbreviated as X
nOr X
mPeptide sequence at least one be that length is at least 20 amino acid, the natural peptide sequence that does not link to each other with hydrophobin, described polypeptide change at least 20 ° of contact angles after bag is by glass surface.
2. the polypeptide of claim 1, wherein said structural formula (I) comprises I class hydrophobin.
3. the polypeptide of claim 1, wherein said structural formula (I) comprises the hydrophobin that is selected from dewA, rodA, sc3, hypA, hypB, basf1, basf2.
4. the polypeptide of claim 1, wherein said structural formula (I) comprise and are selected from SEQ ID NO:2,4,6,8,10,12,14 sequence.
5. the polypeptide of claim 1, wherein X
nOr X
mComprise and be selected from SEQ ID NO:16,18 peptide sequence.
6. the polypeptide of claim 1, wherein X
nOr X
mBe (His)
4-10Sequence.
7. the polypeptide of claim 1, wherein said structural formula (I) comprise and are selected from SEQ ID NO:20,22,24 polypeptide.
8. the nucleic acid of the polypeptide of each definition among the coding claim 1-7.
9. the method for preparing the polypeptide of each definition among the claim 1-7, it is by expressing the nucleic acid of claim 8 definition, and if separate thus obtained protein after being suitably in purifying in host living beings.
10. the method for claim 9, wherein used host living beings is intestinal bacteria.
11. the hydrophobin of each definition is used to wrap the purposes by the surface among the claim 1-7.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102005005737.3 | 2005-02-07 | ||
| DE200510005737 DE102005005737A1 (en) | 2005-02-07 | 2005-02-07 | New hydrophobin polypeptide not connected in normal manner with an external hydrophobin useful for coating of a glass surfaces |
| DE200510007480 DE102005007480A1 (en) | 2005-02-17 | 2005-02-17 | New hydrophobin polypeptide not connected in normal manner with an external hydrophobin useful for coating of a glass surfaces |
| DE102005007480.4 | 2005-02-17 | ||
| PCT/EP2006/050719 WO2006082251A2 (en) | 2005-02-07 | 2006-02-07 | Novel hydrophobin fusion products, production and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101115768A true CN101115768A (en) | 2008-01-30 |
| CN101115768B CN101115768B (en) | 2012-09-05 |
Family
ID=36745955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200680004292XA Expired - Fee Related CN101115768B (en) | 2005-02-07 | 2006-02-07 | Novel hydrophobin fusion products, production and use thereof |
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| Country | Link |
|---|---|
| CN (1) | CN101115768B (en) |
| DE (1) | DE102005005737A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101193911B (en) * | 2005-06-10 | 2013-06-12 | 巴斯福股份公司 | Cysteine-depleted hydrophobin fusion proteins, their production and use thereof |
| CN108004254A (en) * | 2017-12-13 | 2018-05-08 | 天津大学 | The albumen and application of hydrophobin mHGFI genes and expression |
| CN108060169A (en) * | 2017-12-13 | 2018-05-22 | 天津大学 | The albumen and application of hydrophobin mHFBI genes and expression |
| CN111518824A (en) * | 2020-04-29 | 2020-08-11 | 南开大学 | Method for improving exogenous nucleic acid transformation efficiency |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ514891A (en) * | 1999-03-25 | 2003-10-31 | Valtion Teknillinen | Process for partitioning of proteins using hydrophobin proteins fused to the cell or protein of choice |
-
2005
- 2005-02-07 DE DE200510005737 patent/DE102005005737A1/en not_active Withdrawn
-
2006
- 2006-02-07 CN CN200680004292XA patent/CN101115768B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101193911B (en) * | 2005-06-10 | 2013-06-12 | 巴斯福股份公司 | Cysteine-depleted hydrophobin fusion proteins, their production and use thereof |
| CN108004254A (en) * | 2017-12-13 | 2018-05-08 | 天津大学 | The albumen and application of hydrophobin mHGFI genes and expression |
| CN108060169A (en) * | 2017-12-13 | 2018-05-22 | 天津大学 | The albumen and application of hydrophobin mHFBI genes and expression |
| CN108004254B (en) * | 2017-12-13 | 2020-04-17 | 天津大学 | Hydrophobin mHGFI gene, expressed protein and application |
| CN108060169B (en) * | 2017-12-13 | 2020-04-17 | 天津大学 | Hydrophobin mHFBI gene, expressed protein and application |
| CN111518824A (en) * | 2020-04-29 | 2020-08-11 | 南开大学 | Method for improving exogenous nucleic acid transformation efficiency |
| CN111518824B (en) * | 2020-04-29 | 2022-07-26 | 南开大学 | Method for improving exogenous nucleic acid transformation efficiency |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101115768B (en) | 2012-09-05 |
| DE102005005737A1 (en) | 2006-08-17 |
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