WO2005033316A2 - Sekretion von proteinen aus hefen - Google Patents
Sekretion von proteinen aus hefen Download PDFInfo
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- WO2005033316A2 WO2005033316A2 PCT/EP2004/010346 EP2004010346W WO2005033316A2 WO 2005033316 A2 WO2005033316 A2 WO 2005033316A2 EP 2004010346 W EP2004010346 W EP 2004010346W WO 2005033316 A2 WO2005033316 A2 WO 2005033316A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Definitions
- the present invention relates to expression constructs comprising the coding nucleic acid sequence for a shuttle peptide construct processable by yeast cells; corresponding expression vectors containing such constructs; processes carried out with the aid thereof for the recombinant production of target proteins; hosts transformed with it; Shuttle peptides and nucleic acid sequences coding therefor; Nucleic acid sequences coding for such shuttle peptides, fused with a foreign protein; Hydrophobin proteins, which were produced using such shuttle peptides, and the use of hydrophobins for coating objects, such as e.g. Leather.
- yeasts as hosts for heterologous protein expression are widely used. The reason for this is that yeast has several advantages as an expression system. Compared to bacteria and other eukaryotic cells, they can grow in higher density and have the ability for protein glycosylation and post-translational modification. In addition, the products produced and secreted by yeasts can be purified in a simple manner because the yeasts have high resistance to cell lysis and small amounts of foreign protein are usually found in the growth medium. In addition, yeasts can grow faster than other eukaryotic cells in high density on inexpensive nutrient media.
- an expression vector for the expression of heterologous proteins in yeast which, flanked by suitable transcription and translation start or termination sequences, comprises the coding sequence for a hybrid precursor polypeptide which comprises the signal peptide as elements and the leader peptide of a protein secreted by yeasts and a heterologous protein flanked by N-terminal and C-terminal propeptide sequences of the heterologous protein.
- Hydrophobins are small, approximately 100 amino acid residues, cysteine-rich proteins with interesting technical properties. You can make hydrophobic surfaces hydrophilic. They make hydrophilic surfaces hydrophobic.
- hydrophobins from edible mushrooms (cf. SEQ ID NO: 21 and 22).
- WO-A-00/58342 relates to the purification of hydrophobin-containing fusion proteins by phase extraction.
- WO-A-01/57066 describes stabilization, solubilization and the associated better use of hydrophobins by sulfite treatment.
- WO-A-01/57076 describes the purification of hydrophobin by adsorption on Teflon beads and the elution by means of a detergent such as Tween at low temperatures.
- WO-A-01/57528 describes the fixation of hydrophobins on surfaces by the use of tween and temperatures up to 85 degrees Celsius.
- WO-A-01/74864 describes atypical hydrophobins (only one disulfide bridge) with the names RdIA and RdlB (cf. SEQ ID NO: 19 and 20) from filamentous bacteria, in particular Streptomyces sp.
- the hydrophobin is used for the surface treatment of various objects such as windows, contact lenses and vehicle bodies. It is also proposed to produce the proteins described there in a recombinant host that releases the proteins into the medium. After detachment The hydrophobin-containing medium should be suitable for surface coating of the host. Experimental evidence for actual expression and secretion is not provided.
- the object of the present invention is to provide agents which make it possible to secrete expressed homologous or, in particular, heterologous proteins expressed in yeast, in particular Schizosaccharomyces pombe, from the yeast cells into the surrounding medium.
- means should be provided which allow the secretion of recombinantly produced hydrophobin from the host cell.
- Sig signal peptide
- SP shuttle peptide
- hydrophobin DewA protein protein according to SEQ ID NO: 14 with coding sequence according to SEQ ID NO: 13; preprotein with signal sequence: SEQ ID NO: 16 with coding nucleic acid sequence according to SEQ ID NO: 15
- This protein is a class I representative of hydrophobins, i.e. of secreted fungal envelope proteins with the ability to self-assemble.
- the DNA sequence coding for the target protein (DewA) (SEQ ID NO: 13) is at the 3'-terminal end for a peptide pheromone from S. pombe (P factor; amino acid sequence according to SEQ ID NO: 6 for mature P-factor) coding DNA sequence (SEQ ID NO: 5 for mature P-factor) fused.
- the resulting fusion protein contains all the signal sequences necessary for the secretion of the pheromone and the target protein fused to it, in particular the cleavable signal peptide (SEQ ID NO: 4).
- the fusion protein is processed proteolytically.
- the pheromone (P factor) (SEQ ID NO: 6) and the target protein (hydrophobin; SEQ ID NO: 14) are secreted separately into the medium.
- the finding according to the invention is surprising in that the regulatory elements of the P-factor preprotein (N-terminal to the mature pheromone) are obviously not sufficient to control the secretion of the target protein by the yeast cells. Only the use of a construct in which an additional, co-secreting protein component (the mature pheromone) is processably connected upstream of the target protein to be secreted enables the desired secretion of the target protein into the culture medium.
- secrete is a protein which is expressed intracellularly by a host cell, in particular of yeasts, and is excreted from the cell, preferably into the surrounding medium, through the cell membrane via the cell's own mechanisms.
- Protein precursor in its originally expressed form, such as a preprotein, with N- and / or C-terminal peptide sequences that are no longer present in the mature processed protein
- proteolytic processes in and / or outside the host cell.
- a "processable linkage" is given when individual protein sections in a protein to be processed are linked via peptide bonds which can be cleaved by a proteolytic enzyme of the host cell.
- the “processing” can take place N-terminally and optionally also C-terminally to the sequence of the mature, processed protein (target protein).
- target protein A “homologous” target protein, although originally expressed in the host used according to the invention, is therefore a host's own protein, but is secreted by the host cells due to the transformation of the host with an expression construct according to the invention.
- a “heterologous” target protein is not originally expressed in the host used according to the invention, is therefore not a host protein, but is secreted by the host cells due to the transformation of the host with the expression construct according to the invention.
- a “shuttle peptide” is part of a “shuttle peptide construct” that can be processed in the host cell used according to the invention. Together with one or more processable regulative C- and / or N-terminal, preferably N-terminal, associated peptide fragments, such as signal sequences, leader sequences, it forms the shuttle peptide construct.
- the shuttle peptide is e.g. to the signal peptide a polypeptide secreted by the host cell.
- the regulatory elements are preferably processed intracellularly.
- the shuttle peptide can also be secreted if it is fused, preferably C-terminally, to a target protein in a processable manner. This C-terminal processing is preferably carried out, i.e. Cleavage of the target protein, proteolytic during secretion, e.g. during passage through the host cell envelope or in extracellular space, e.g. in the surrounding culture medium, by the cell's own proteases.
- An “expression construct” or an “expression cassette” according to the present invention comprises, operatively linked, to the coding nucleic acid sequence of a processable shuttle peptide construct according to the above definition, the start and termination signals necessary for controlling expression in a special host system, such as in particular yeast cells Transcription and, if necessary, translation.
- the expression construct in particular comprises binding sites for transcription factors. 5'- Upstream of the coding sequence is a constitutive or inducible, native or heterologous, natural or synthetic promoter operable in the host cell.
- the expression construct also includes a number of restriction enzyme sites, e.g. B. those for inserting the construct into an expression vector.
- the expression construct can include a gene for a selectable marker.
- An “expression vector” describes a construct that can be obtained by inserting an expression cassette according to the invention into a replicon, such as, for example, into a plasmid, cosmid or a virus.
- a replicon such as, for example, into a plasmid, cosmid or a virus.
- Such a vector is capable of autonomous replication or integration into the host genome and contains the necessary ones Control sequences for controlling transcription and optionally translation of the coding nucleic acid sequences according to the invention for a processable shuttle peptide construct as defined above.
- a first subject of the invention relates to an expression construct comprising the coding nucleic acid sequence for a shuttle peptide construct of the general formula which can be processed by yeast cells
- telomere sequences for a) a signal peptide (Sig) in the 5'-3 'direction, processably linked to b) at least one shuttle peptide (SP) that can be secreted by the yeast cells; and optionally one or more nucleic acid sequences which promote processing and / or secretion, 5 'or 3' terminal to the coding signal peptide sequence.
- Sig signal peptide
- SP shuttle peptide
- the coding sequences for SP and Sig are in the same reading frame and, in addition, a processable sequence between the C-terminus of Sig and the N-terminus of SP is formed during translation.
- This processable sequence can be, for example, an artificially introduced, proteolytically cleavable natural or synthetic adapter sequence. However, this is preferably part of the C-terminus of Sig or N-terminus of SP.
- the adapter sequence can be processed in such a way that the split sequence can be found in whole or in part at the C-terminus of Sig or the N-terminus of SP. The latter is possible as long as this does not significantly impair, in particular not prevent, the ability to secrete SP.
- the invention relates to such expression constructs which code for a processable shuttle peptide construct which is derived from a polypeptide which is processed by yeasts in the broadest sense.
- yeasts selected from Ascomycetes.
- Preferred yeasts are selected from those of the Class of the Archiascomycetes, the order of the Schizosaccharomycetales and particularly preferably selected from yeasts of the genus Schizosaccharomyces, such as S. pombe.
- the processable shuttle peptide construct is derived in particular from a pheromone preprotein from a yeast, the pheromone being produced from the preprotein by N- and C-terminal processing.
- the pheromone N-terminal preferably has a polypeptide which can be split off by processing and which in particular comprises the elements required for processing and / or secretion of the preprotein, such as signal peptide and possibly leader peptide, and the required protease interfaces.
- Mushroom pheromones are known and e.g. described both for basidiomycetes such as Ustilago maydis (Urban, M., Kahmann, R. and Bolker, M. (1996)
- Three subfamilies of pheromone and reeeptor genes generate mutiple B mating speeifities in the mushroom Coprineus cinereus (Genetics 154 ( 3): 1115-1123)) and ascomycetes such as Schizosaccharomyces pombe (Imai, Y.
- Pheromones suitable according to the invention are relatively small peptides (such as 5 to 40 or 8 to 30 amino acids). They usually do not show significant homology in the primary sequence. They are formed as preproteins, processed proteolytically and released into the culture medium.
- pheromones or corresponding pre-proteins examples include the so-called P and M factors or their pre-proteins from S. pombe. (cf.Imai, Y. and Yamamoto, M. (1995) The fission yeast mating pheromone P-factor: its molecular strueture, gene strueture, and ability to induce gene expression and G1 arrest in the mating partner (Genes Dev 8 (3 ): 328-338), Davey, J.
- the P-factor preprotein has, for example, a DNA sequence according to SEQ ID NO: 9 and a protein sequence according to SEQ ID NO: 10.
- the preprotein comprises an N-terminal signal peptide sequence bridged with four successive pheromone peptide sequences which can be separated by processing (cf. FIG. 3).
- the processable shuttle peptide construct is designed such that it contains a signal polypeptide (Sig) which is processably linked to the N-terminal end of a C-terminally processable pheromone polypeptide (Pher).
- Sig signal polypeptide
- Pher C-terminally processable pheromone polypeptide
- the signal polypeptide comprises or is identical to the proteolytically cleavable native signal polypeptide (e.g. SEQ ID NO: 4 encoded by SEQ ID NO: 3) of the pheromone preprotein.
- the C-terminal processed pheromone polypeptide comprises a C-terminal protease interface.
- the expression construct preferably further comprises the coding nucleic acid sequence for a homologous or heterologous target protein (Targ), processably linked to the C-terminus of the shuttle peptide construct (Sig-SP).
- the invention preferably relates to expression constructs of the type described above, comprising the coding nucleic acid sequence for a fusion protein of the general formula which can be processed by yeast cells
- L1 and L2 stand for processable linkers or adapter sequences and n and m stand independently for 0 or 1. However, n is preferably 1 and m is O.
- L1 and L2 can be natural or synthetic linkers. They comprise at least one proteolytically processable peptide sequence. If necessary, with L1 and / or L2, e.g. the processing, secreting, transcription and / or translation-promoting effector functions are associated.
- the coding nucleic acid sequence for the processable shuttle peptide construct being a sequence coding for a signal polypeptide (Sig) according to SEQ ID NO: 3 or a functional equivalent thereof, operatively linked to that for the mature P factor pheromone (Pher) encoding nucleic acid sequence according to SEQ ID NO: 5 or a functional equivalent thereof.
- Sig signal polypeptide
- Pher mature P factor pheromone
- the linker L2 is preferably not present.
- the linker L1 is preferably provided and comprises the coding sequence for a polypeptide according to amino acid residues 21 to 30 in SEQ ID NO: 10.
- L1 bridges the signal polypeptide with the first pheromone building block (positions 31 to 57 in SEQ ID NO : 10) the prehormone.
- the C-terminal end of L1 corresponds to a recognition sequence of the protease required for the proteolytic processing.
- the coding nucleic acid sequence for the processable shuttle peptide construct comprises a sequence according to SEQ ID NO: 1.
- the same procedure can also be used with the help of the M factor - the second pheromone occurring in S. pombe - and applicable to the expression of any homo- and heterologous target proteins (target proteins).
- Genomically there are three genes (mfmf, SEQ ID NO: 42; mfm2 SEQ ID NO: 45; and mfm3 + , SEQ ID NO: 48) which each encode the M factor, the pheromone of the cells with the minus pairing type.
- a preprotein (SEQ ID NO: 43, 46 and 49) is first formed from each gene, which is processed as part of the secretion.
- the M factor (YTPKVPYMC; SEQ ID NO: 51), encoded by SEQ ID NO: 44, 47 and 50) is released into the medium as a mature pheromone (cf. FIG. 9).
- shuttle peptide constructs suitable according to the invention could therefore be derived from the coding sequences according to SEQ ID NO: 42, 45 or 48, which code for M factor signal peptide, functionally linked to an M factor pheromone.
- Non-limiting examples of corresponding coding shuttle peptide sequences include e.g.
- Functional equivalents can contain the sequence segments located 5 'upstream from the coding sequence of the mature M factor (SEQ ID NO: 44, 47 or 50) unchanged or modified (e.g. by deletion of one or more nucleic acid residues), and thus for an in encode its amino acid sequence modified shuttle peptide, which functionally links the mature M factor peptide sequence with a, eg C-terminal shortened signal sequence section includes.
- a target protein (target) expressed according to the invention can be derived from any prokaryotic or eukaryotic organism, in particular humans, animals or yeasts, as long as it can be expressed by the host cell in the manner according to the invention as a component of a fusion protein with the shuttle peptide (SP) and is processable.
- the secreted and processed product can be therapeutically useful or have other advantageous application properties.
- therapeutically useful proteins are immunoglobulins, Peptide hormones, growth factors, lymphokines, protease inhibitors and the like. Hydrophobins are particularly worthy of mention as an example of target proteins with other properties which are of interest in terms of application technology.
- the target protein is a hydrophobin, in particular a hydrophobin of class I.
- hydrophobins are relatively small (100 + 25 amino acids) moderately hydrophobic proteins with a conserved motif of 8 cysteines (X -CX 2-38 5- 9-CCX 11-39 -CX 8- 23 -CX 5-9 -CCX. 6 18 -CX 2- ⁇ 3 ). Hydrophobins can assemble at hydrophilic-hydrophobic interfaces to protein films. Such aggregates of class I hydrophobins are insoluble in SDS, while aggregates of class II hydrophobins are soluble in SDS (Wessels, JGH (1997) Hydrophobins: Proteins that change the nature of the fungal surface. Adv Microb Physiol 38: 1-45) ,
- Hydrophobins which can be used according to the invention are derived in particular from fungi, e.g. from Ascomycetes, such as those of the genus Aspergillus, in particular A. nidulans.
- Useful hydrophobins are also known from the prior art mentioned above and are not restricted to those from fungi.
- Non-limiting examples of useful hydrophobins are selected from SEQ ID NO: 14 (DewA), SEQ ID NO: 19 (RdIA) SEQ ID NO: 20 (RdlB) SEQ ID NO: 21 (HYP1) SEQ ID NO: 22 (HYP4) and SEQ ID NO: 56 (RodA).
- the RodA protein together with the DewA protein is part of the outer spore shell of A. nidulans.
- the invention also relates to expression vectors comprising, in operative linkage with at least one regulatory nucleic acid sequence, an expression construct as defined above.
- the invention also relates to recombinant microorganisms which contain, if appropriate stably integrated into the host genome, at least one expression vector or an expression construct as defined above.
- a “recombinant microorganism in the sense of the present invention comprises at least one expression vector according to the invention or an expression construct according to the invention and is derived from yeasts in the broadest sense.
- the yeasts are derived from Ascomycetes.
- Preferred yeasts are selected from the class of the Archiascomycetes, the order of the Schizosaccharomycetales, and particularly preferably selected from yeasts of the genus Schizosaccharomyces, such as S. pombe.
- Another object of the invention relates to shuttle peptide constructs that can be processed by yeast cells, derived from a pheromone preprotein from a yeast, the pheromone being derivable and secrete from the preprotein by N- and C-terminal processing.
- Preferred shuttle peptide constructs containing a signal polypeptide N-terminai are processably linked to the C-terminally processed pheromone polypeptide.
- the signal polypeptide is preferably the proteolytically cleavable native signal polypeptide of the pheromone preprotein and the C-terminally processed pheromone polypeptide comprises the C-terminal protease interface.
- Preferred shuttle peptide constructs are derived from pheromone preproteins, from yeasts, in particular preproteins of the factors P and M from S. pombe.
- Particularly preferred shuttle peptides comprise an amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof.
- Another object of the invention relates to a method for the recombinant production of a target protein, wherein a recombinant microorganism according to the above definition is cultivated, which expresses the nucleic acid sequence encoding the target protein and the target protein secreted into the culture medium, such as e.g. a hydrophobin as defined above, isolated.
- the invention further relates to nucleic acids coding for a shuttle peptide construct as defined above; as well as nucleic acids coding for a fusion protein which can be processed by yeast lines and comprises a target protein and as defined above.
- the invention also relates to hydrophobins obtainable by a process according to the invention.
- the invention relates to the use of such a hydrophobin for surface treatment, in particular the surface of objects selected from glass, fibers, fabrics, leather, lacquered objects, such as e.g. Motor vehicle bodies, foils, facades treated.
- the invention also relates to the use of hydrophobins for the surface treatment of fibers, fabrics and leather.
- polypeptide also included according to the invention are “functional equivalents” of the specifically disclosed or used polypeptide / proteins. This applies both to the intermediately formed fusion proteins and to their components, ie target proteins (target), shuttle peptides (SP) such as pheromones (Pher) also for signal peptides (Sig) and linkers.
- target target proteins
- SP shuttle peptides
- Sig signal peptides
- linkers linkers.
- polypeptide is used as a generic term for polypeptide / protein.
- “Functional equivalents” or analogs of the specifically disclosed polypeptides are, within the scope of the present invention, different polypeptides thereof which continue to have the desired biological activity.
- Analog shuttle peptides should continue to be suitable for controlling the secretion and processing of the target protein.
- the functional equivalents should also correspond accordingly of components of the shuttle peptide, such as signal polypeptide, pheromone, linker, furthermore have the properties required for effective secretion and processing of the fusion protein with release of the target protein.
- “Functional equivalents” of polypeptides according to the invention can in particular contain residues of natural linker or adapter sequences which are formed by proteolytic cleavage at the C- and / or N-terminal.
- “functional equivalents” are understood to mean, in particular, mutant proteins which have at least one of the sequence positions of the above-mentioned concrete sequences different from the specifically mentioned amino acid, but nevertheless have one of the above-mentioned biological activities.
- “Functional equivalents” thus include the mutant proteins obtainable by one or more amino acid additions, substitutions (cf. examples in the following table), deletions and / or inversions, the changes mentioned being able to occur in any sequence position as long as they exist a mutant protein with the property profile according to the invention.
- Suitable residues for amino acid substitutions include:
- Functional equivalence is particularly given when the activity pattern between mutant and unchanged polypeptide matches qualitatively.
- modified shuttle peptides express or secrete the same target protein with higher or lower efficiency in the same host; or that modified target proteins have an increased or decreased pharmacological effect or modified application properties.
- “Functional equivalents” in the above sense also include precursors of the polypeptides described and functional derivatives and salts of the polypeptides.
- salts means both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention.
- Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts, such as, for example, sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, such as, for example, amines, such as triethanolamine, arginine , Lysine, piperidine and the like.
- Acid addition salts such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are also a subject of the invention.
- “Functional derivatives” of polypeptides according to the invention can also be prepared on functional amino acid side groups or on their N- or C-terminal end using known techniques.
- Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine, N-acyl derivatives of free amino groups, produced by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.
- “Functional equivalents” naturally also include polypeptides which are accessible from organisms other than those specifically mentioned, and naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.
- “Functional equivalents” also include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.
- “Functional equivalents” are also fusion proteins which contain one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without mutual substantial functional impairment of the fusion protein parts).
- heterologous sequences are signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.
- “Functional equivalents” encompassed according to the invention are homologs to the specifically named polypeptides. These have at least 60%, preferably at least 75%, in particular at least 85%, such as 90%, 95% or 99%, homology to one of the concretely disclosed Sequences calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sei. (USA) 85 (8), 1988, 2444-2448.
- a percentage homology of a homologous polypeptide according to the invention means in particular percentage identity of the amino acid residues based on the total length of one of the amino acid sequences specifically described herein.
- equivalents according to the invention include polypeptides in deglycosylated or glycosylated form and also modified forms obtainable by changing the glycosylation pattern.
- Homologs of the proteins or polypeptides according to the invention can be generated in a manner known per se by mutagenesis, e.g. by point mutation or shortening of the protein.
- Nucleic acid sequences according to the invention in particular those which code for one of the above polypeptides and their functional equivalents, comprise single and double-stranded DNA and RNA sequences, such as e.g. also cDNA and mRNA.
- nucleic acid sequences mentioned here are either of natural origin or can be prepared in a manner known per se by chemical synthesis from nucleotide components, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid components.
- oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
- the attachment of synthetic oligonucleotides and the filling of gaps with the aid of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning methods are described in Sambrook et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
- the invention also relates to nucleic acid sequences coding for one of the above polypeptides and their functional equivalents, which e.g. are accessible using artificial nucleotide analogs.
- the invention relates both to isolated nucleic acid molecules which code for polypeptides according to the invention or biologically active sections thereof, and to nucleic acid fragments which e.g. are suitable for use as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.
- nucleic acid molecules according to the invention can also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region.
- nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and, moreover, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals be when it's chemically synthesized.
- a nucleic acid molecule according to the invention can be isolated using standard molecular biological techniques and the sequence information provided according to the invention.
- cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
- nucleic acid molecule comprising one of the disclosed sequences or a portion thereof can be isolated by polymerase chain reaction using the oligonucleotide primers which have been created on the basis of this sequence.
- the nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis.
- the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.
- nucleotide sequences mentioned enable the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms.
- probes or primers usually comprise a nucleotide sequence region which, under stringent conditions, can be attached to at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand are hybridized.
- nucleic acid sequences according to the invention are derived from the specifically disclosed sequences and differ from them by addition, substitution, insertion or deletion of one or more nucleotides, but continue to code for polypeptides with the desired property profile.
- nucleic acid sequences which comprise so-called silent mutations or which are modified in accordance with the codon usage of a specific source or host organism, in comparison with a specifically named sequence, as well as naturally occurring variants, such as e.g. Splice variants or allele variants, thereof.
- Sequences obtainable by conservative nucleotide substitutions i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility are also a subject of the subject.
- the invention also relates to the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population due to natural variation. These natural variations usually cause a 1 to 5% variance in the nucleotide sequence of a gene.
- the invention also encompasses nucleic acid sequences which hybridize with the abovementioned coding sequences or are complementary thereto.
- These polynucleotides can be found when screening genomic or cDNA banks and, if appropriate, can be amplified therefrom using suitable primers by means of PCR and then isolated, for example, using suitable probes.
- the property of being able to “hybridize” to polynucleotides means the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions non-specific bindings between non-complementary partners are avoided.
- sequences should be closed 70-100%, preferably 90-100%, are complementary
- the property of complementary sequences to be able to bind specifically to one another is demonstrated, for example, in the Northern or Southern blot technique or in primer binding in PCR or RT-PCR Usually oligonucleotides with a length of 30 base pairs or more are used for this purpose.
- Stringent conditions mean, for example in Northern blot technology, the use of a washing solution, for example 50-70 ° C., preferably 60-65 ° C. 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCI, 0.3M Na citrate, pH 7.0) for the non-specific hybrid elution cDNA probes or oligonucleotides.
- the invention also relates to expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide to be expressed according to the invention; and vectors comprising at least one of these expression constructs.
- Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence.
- An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
- sequences which can be linked operatively are targeting sequences and enhancers, polyadenylation signals and the like.
- Other regulatory elements include selectable markers, amplification signals, origins of replication and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- the coding nucleic acid sequences can be contained in one or more copies in the gene construct.
- yeast promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, nmt1, nmt41 and nmt81.
- Suitable promoters for the yeast S. pombe are e.g. to name: nmtl, nmt41, nmt81, adhl, fbpl, SV40 or CaMV. Further information at (http://pinqu.salk.edU/ ⁇ forsburq/vectors.html#exp).
- the promoters differ in their transcription rate. The selection depends on the desired level of expression. The same applies to other yeasts.
- Suitable yeast promoters are described, for example, in published US patent application 2003/0077831, which is hereby expressly incorporated by reference.
- inducible promoters such as e.g. light-inducible and in particular temperature-inducible promoters.
- the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
- the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it.
- the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
- an increase in translation is also possible, for example, by improving the stability of the mRNA.
- An expression cassette is produced by fusing a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Common recombination and cloning techniques are used, such as those described in T. Maniatis, EF Fritsch and J.
- the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
- Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouweis P.H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
- vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
- Constructs suitable for the yeast S. pombe can be mentioned as examples of expression vectors suitable according to the invention (see, for example: (http://pinqu.salk.edU/ ⁇ forsburq/vectors.html#exp).
- REP1, REP3, REP4 (Maundrell, K. (1990). Nmtl of fission yeast: a highly transcribed gene completely repressed by thiarnine. J. Biol. Chem. 265: 10857-10864) REP41, REP42, REP81, REP82 (Basi, G., Schmid, E. and Maundrell, K. (1993) TATA box mutations in the Schizosaccharomyces pombe nmtl promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123: 131-136)
- Yeast expression vectors for expression in yeast S. cerevisiae such as pYEpSed (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).
- Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.
- recombinant microorganisms can be produced which, for example, have been transformed with at least one vector according to the invention and can be used to produce the polypeptides according to the invention.
- the recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system.
- Common cloning and transfection methods known to the person skilled in the art such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
- all organisms which enable expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives are suitable as host organisms.
- Preferred host organisms are yeasts.
- Auxotrophic markers are often used in yeasts to select transformants.
- the strain to be transformed lacks a protein which is necessary for the production of certain metabolic products.
- the corresponding active protein is introduced into the cell by the vector used. Commonly used markers are
- Genes e.g. uracil, leucine, histidine or tryptophan biosynthesis.
- Successfully transformed organisms can be selected using marker genes, which are also contained in the vector or in the expression cassette.
- marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting.
- Microorganisms which have been successfully transformed with a vector and carry an appropriate antibiotic resistance gene e.g. G418 or hygromycin
- an appropriate antibiotic resistance gene e.g. G418 or hygromycin
- Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.
- the combination of the host organisms and the vectors which match the organisms, such as plasmids, viruses or phages, such as, for example, plasmids with the RNA polymerase / promoter system, the phages 8 or: or other temperate phages or transposons and / or further advantageous regulatory ones Sequences form an expression system.
- the invention further relates to methods for the recombinant production of a target protein as defined above.
- the recombinant microorganism can be cultivated and fermented by known methods.
- suitable cultivation conditions for example for S. pombe in Alfa et al. (Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of Genetics 1, pp 395-446, Plenum Press, New York) or for S. cerevisiae in Kaiser et al. (Kaiser, C, Michaelis, S. and Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, New York).
- the cells are separated from this and the target protein is obtained from the supernatant by known protein isolation methods.
- Purification of the target protein can be achieved with known chromatographic methods, such as molecular sieve chromatography (gel filtration), ion exchange chromatography, such as Q-Sepharose chromatography, and hydrophobic chromatography, as well as with other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemical Working Methods, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
- vector systems can also be used which code for modified polypeptides or fusion proteins which serve for easier purification.
- suitable modifications are, for example, so-called “tags” which act as anchors, such as, for example, the modification known as a hexa-histidine anchor or epitopes which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D ., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press).
- anchors can be used to attach the proteins to a solid support, such as, for example, a polymer matrix, which can be filled, for example, in a chromatography column, or can be used on a microtiter plate or on another support.
- a solid support such as, for example, a polymer matrix, which can be filled, for example, in a chromatography column, or can be used on a microtiter plate or on another support.
- these anchors can also be used to recognize the proteins.
- customary markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors, can be used to derivatize the proteins.
- WO-A-01/57066 which describes the stabilization, solubilization and the associated better use of hydrophobins by sulfite treatment
- WO-A-01/57076 which describes the purification of Hydrophobin by adsorption on Teflon beads and elution with detergent such as Tween describes at low temperatures
- WO-A-01/57528 which describes the fixation of hydrophobins on surfaces by the use of Tween and temperatures up to 85 degrees Celsius
- solid materials such as glass, fibers, fabrics, leather, lacquered objects, foils, facades, can be coated with hydrophobin.
- Figure 1 different constructs according to the invention for the secretion of the hydrophobins from S. pombe.
- FIG. 2A shows the genomic sequence of the DewA gene (SEQ ID NO: 39); the sequences of the two introns are underlined; B) the amino acid sequence and in brackets the corresponding DNA sequence of the DewA protein from Aspergillus nidulans; the signal sequence is printed in bold, the partial sequence following the signal sequence corresponds to the sequence of mature DewA; C) the amino acid sequence and in brackets the corresponding DNA sequence of the HA tag.
- 3A the amino acid sequence and, in parentheses, the corresponding DNA sequence of the P-factor preprotein; the signal sequence is printed in bold; the underlined partial sequences following the signal sequence correspond to the sequences of the four maternal pheromone peptides; the pheromone closest to the signal peptide is called the P factor; B) amino acid sequence and in brackets the corresponding DNA sequence of the cleavable signal peptide and the subsequent 6 amino acids (underlined) of the P-factor preprotein; C) amino acid sequence and in brackets the corresponding DNA sequence for the "P-Shuttle" according to the invention; the signal sequence is printed in bold; the underlined sub-sequence following the signal sequence corresponds to the sequence of the material P factor;
- FIG. 4 shows a fusion protein according to the invention consisting of the “P-shuttle” sequence (signal sequence bold; mature P protein underlined), the mature DewA (double underlined) and the C-terminally fused HA tag (SEQ ID NO: 18; coded) from SEQ ID NO: 17);
- FIG. 5 shows the immunological detection of hydrophobins in S. pombe *
- Detection fused with an HA tag cloned into the expression vector pJR1-3XL and transformed into S. pombe. "Membrane fraction” and “cytosolic proteins” were separated by SDS-PAGE. The detection in the Westem analysis was carried out with HA antibodies. The size standard in kDa is given on the left.
- Figure 6 shows the immunological detection of the expression of hydrophobins in S. pombe.
- the PDewAHA protein was expressed in S ⁇ pombe.
- the cells were harvested, the culture supernatant was aliquoted and part of the TCA was precipitated.
- the protein was detected by SDS-PAGE and Western blot with the help of HA antibodies.
- the bands marked with * correspond to the precursor protein (approx. 18 kD, upper band) and the mature form (approx. 17 kD lower band).
- S. pombe cells were transformed with plasmids which express P + 6DewA by a strong promoter (pJR1-3XL) or weaker promoter (pJR1-81XL).
- the cells carry a version of the prp7 gene chromosomally with a c-myc tag. This serves as a control to rule out that the culture supernatant has been contaminated by lysed cells.
- Cells were harvested (pellet), the culture supernatant TCA- precipitated (supernatant).
- the proteins were detected by SDS-PAGE and Western blot with the help of antibodies against HA (A) or against c-myc (B).
- Figure 8 shows the detection of secretion using the "P-Shuttle” method.
- S. pombe cells were transformed with plasmids which express PfakDewA through a weaker promoter (pJR1-81XL). The cells were harvested (pellet), the culture supernatant TCA-precipitated (US). The protein was detected according to SDS-PAGE and Westem blot with the help of antibodies against HA.
- FIG. 9 the three genes which each encode the M factor (SEQ ID NO: 51 for mature factor) from S. pombe: A) sequences for the mfm1 + ' gene; B) sequences for the tr7t / 772 + " gene; and C) sequences for the mfm3 * ' gene.
- Figure 10 shows the RodA gene.
- the genomic sequence (SEQ ID NO: 52) of the RodA gene contains two introns (underlined), which are not present in the corresponding coding ORF (SEQ ID NO: 53).
- the preprotein (SEQ ID NO: 54) contains a cleavable signal sequence (printed in bold) which is missing in the mature protein (SEQ ID NO: 56; encoded by SEQ ID NO: 55).
- the cloning steps carried out in the context of the present invention e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of
- DNA was purified from reaction mixtures or after gel electrophoresis using the NucleoSpin Extract Kit (Machery-Nagel, Düren) and plasmid DNA from E. coli was isolated using the NucleoSpin Plasmid Quick Pure Kit (Machery-Nagel, Düren) the manufacturer's instructions.
- Transformations in E. coli were carried out by electroporation using the Gene Pulser II device (BIO-RAD, Kunststoff) using 2 mm electroporation cuvettes (Biozym Diagnostik, Hess. Oldendorf) according to the manufacturer. Transformants were selected on LB medium (150 mg / l) containing ampicillin (Lennox, 1955, Virology, 1: 190).
- PCR amplifications were carried out using the Combizyme DNA polymerase (Invitek, Berlin, Germany) according to the manufacturer's instructions. 100 ⁇ l of reaction volume per 1 pmol of the corresponding primers were used.
- S. pombe The cultivation of S. pombe was carried out as in Alfa et al. (Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of Genetics 1, pp 395-446, Plenum Press, New York).
- cells were harvested by centrifugation at 3,500xg for 5 min.
- the cell pellets were resuspended in 1 ml of 1xPBS and 1 volume of glass beads was added. The mixture was vortexed for 5 min, the supernatant removed over the glass beads.
- strains DH5 ⁇ (Invitrogen), XL10-Gold (Stratagene) or BL21 (BioLabs) were used to work with E. coli.
- S. pombe strains used are from the gap yeast strain collection of the working group of Prof. Dr. G. Rodel taken from the Institute of Genetics at the Technical University of Dresden.
- Example 1 Production of the expression construct DewA and DewAHA and cloning into the vector pJR1-3XL
- the reaction mixture was separated by gel electrophoresis and the corresponding DNA band eluted as described above.
- the fragment which is flanked on both sides by a Sa / ⁇ HI site, which was inserted through the primer, was cut with the restriction endonuclease ßamHI (Invitrogen) according to the manufacturer's instructions and purified from the reaction mixture (see above).
- the vector pUC18 (Yanisch-Pron, C, Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33: 103) was also cut with ßamHI, separated by gel electrophoresis and then eluted from the gel (see above).
- Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli.
- Recombinant plasmids were identified after plasmid preparation and subsequent restriction digestion. After cloning, the correct DNA sequence of the cloned PCR products was - as with all constructs produced below - verified by sequencing. Sequencing reactions were carried out according to Sanger et al. (Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc NatI Acad Sei USA 74: 5463-5467).
- the sequencing reactions were carried out using the "Thermo-Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP" (Amesham Pharamacia Biotech, Freiburg) and S'-side IRD800 labeled primers (MWG Biotech AG, Ebersberg). The products were separated and the sequence evaluated using the automatic LI-COR 4000/4200 (MWG Biotech AG, Ebersberg) sequencing system.
- a construct that contains the intron-containing genomic DewA gene cloned into the ßamHI site of the vector pUC18 was called pDewAgen.
- the intronless ORF was amplified with the subfragments as a template and the distal primers ScDewBamfor and SpDewBamrev.
- the approximately 410 bp long PCR product was separated by gel electrophoresis, purified and cut with the restriction endonuclease ßamHI. Appropriate interfaces had been inserted through the distal primers.
- the cut fragment was purified and cloned into the vector pUC18, also cut with ßamHI. Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli.
- DEPA Since no specific antibodies against DewA are available, DEPA was fused with the HA epitope by OEP to detect heterologous expression.
- the primer pairs SpDewXhofor / DewAHArev and DewAHAfor / DewAHANcorev were used in the primary PCRs.
- DNA of the construct pDewAgen was used as a template for the production of DewHA (+ introns) and DNA of the construct pDewA-ORF for the production of DewHA (-ntrons).
- the vector yEP351 HA (Kettner, K., Friederichs, S., Schlapp, T. and Rodel G (2001) Expression of a VEGF-like) carrying the DNA sequence of the HA tag was used as a template for the PCR with DewAHAfor / DewAHANcorev protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Protein Expr Purif Aug; 22 (3): 479-83). In the final PCRs using the primer pair
- the DNA coding for the HA epitope was fused to the respective DewA DNA.
- the fragments amplified in this way are flanked on the 5 'side by an Xho ⁇ restriction site and on the 3' side by a ⁇ / col restriction site which were introduced with the aid of the distal primers.
- the fragments were separated by gel electrophoresis, purified and cut with the restriction endonucleases Xho ⁇ and Ncol and purified from the reaction mixture.
- the vector pJR1-3XL (Moreno, MB, Duran, A.
- the vectors DewA-HA (+ introns) and DewA-HA (introns) obtained according to a) and b) were in the S. pombe host strain K0103 (t? S ade6-M210 leu1-32 his7-366) as by Schiestl and Gietz (Schiestl, RH and Gietz, RD (1989) High efficency transformation of intact yeast cells using Single stranded nucleic acids as a carrier. Curr Genet 16: 339-346), which are described by the fe ⁇ / 7-32 mutation Conditional leucine auxotrophy of the S. pombe strain is complemented by the LE1 / 2 gene from S. cerevisiae present on the expression vectors. Transformants can thus be selected on minimal medium without leucine. The expression of the fusion proteins in corresponding yeast transformants was determined by means of Western blotting. Analyzes examined.
- anti-HA article 1 583816, anti-HA (12CA5) -mouse monoclonal antibody
- anti-c-myc article 1 667 149, anti-c-myc antibody
- FIG. 5A shows samples of a culture with the insert-free vector (pJR1-3XL, negative control), with an HA-tagged control protein (positive control) and with a vector which contains the HA-tagged DewA gene with introns (DewA -HA (+ introns)).
- samples are culture with the vector (pJR1-3XL, negative control), with a vector which contains the HA-tagged DewA gene without introns (DewA-HA (introns)) or the HA-tagged RodA gene with introns (Rod-AHA ( + lntrons)) contains, applied.
- RodAHA (+ introns) was produced in analogy to the information in Examples 1a) and 1b).
- RodA is another hydrophobin from A. nidulans.
- Example 2 Production of expression vectors for the secretion of the expressed DewA vector containing the construct PDewAHA
- the authentic secretion signal of the A. nidulans protein which is not effective in the split yeast, was first replaced by the cleavable signal peptide of the P factor from S. pombe.
- the P factor is secreted by the cells into the medium as a peptide pheromone. It is synthesized in the cell as a precursor protein (preprotein) consisting of a cleavable N-terminal signal sequence and four P-factor copies, each separated by short spacer sequences, and matures during secretion, including the cleavage of the signal sequence and the proteolytic release of the four P-factor peptides.
- preprotein precursor protein
- the P-factor signal sequence was amplified by means of PCR and genomic DNA from S. pombe as a template using the primer pair SigPXhofor / PDewArev and the corresponding PCR product was purified.
- the PDewAHA fragment was cut with the restriction endonucleases Xho ⁇ and ⁇ / col, separated by gel electrophoresis and ligated into the vector pJR1-3XL (see above) cut with the same restriction endonucleases.
- Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli by electroporation.
- Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing.
- the construct obtained was called PDewAHA.
- the PDewAHA protein was expressed in S. pombe.
- the cells were harvested, the culture supernatant was aliquoted and part of the TCA was precipitated.
- the TCA precipitate was taken up in Laemmli buffer (Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (Nature 227: 680-685)). Cell pellet, supernatant and TCA-precipitated supernatant were examined.
- the protein was detected by SDS-PAGE and Westem blot with the help of HA antibodies. The result is shown in FIG. 6.
- the bands marked with * correspond the precursor protein (approx. 18 kD, upper band) and the mature form (approx. 17 kD lower band).
- Example 3 Production of expression vectors for the secretion of the expressed DewA vector containing the construct P + 6DewAHA
- the sequence of the signal peptide in the fusion protein was determined using OEP using the primer pairs SigPXhofor / P + 6DewArev and P + 6DewAfor / DewAHANcorev with DNA of the construct PDewAHA as template in the primary PCR reactions
- the P + 6DewA fragment was cut with the restriction endonucleases Xho ⁇ and ⁇ / col, separated by gel electrophoresis and in those with the same restriction Sendonucleases cut vector pJR1-3XL (see above) and the ligation mixture transformed by electroporation in E. coli. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing.
- the P + 6DewA fragment was also cloned into the vector pJR-81XL.
- the transcription of the fusion gene is under the control of the weak nmt ⁇ promoter. With this construct, a negative influence of the very high transcription in pJR1-3XL constants on the secretion should be tested.
- Example 2a The experiment was carried out in analogy to Example 2a.
- the amplified sequences are cloned in pJR1-3XI analogously to Example 2a.
- S. pombe cells were transformed with the two plasmids, which express P + 6DewA by a strong promoter (pJR1-3XL) or weaker promoter (pJR1-81XL).
- the cells carry a version of the prpl gene chromosomally with a c-myc tag. This serves as a control to rule out that the culture supernatant has been contaminated by lysed cells.
- Cells were harvested (pellet), the culture supernatant TCA-precipitated (supernatant). The precipitate was taken up in Laemmli buffer and also analyzed.
- the proteins were detected by SDS-PAGE and Western blot with the aid of antibodies against HA (FIG. 7A) or against c-myc (Röche Diagnostics) (FIG. 7B).
- Example 4 Production of expression vectors for the secretion of the expressed DewA vector containing the construct PfakDewAHA
- PCR fragments obtained were separated by gel electrophoresis, purified and used as a template for the final PCR using the primer pair SigPXhofor / DewAHANcorev.
- the PfakDewA fragment thus obtained was cut with the restriction endonucleases Xho ⁇ and ⁇ / col, separated by gel electrophoresis and ligated into the vector pJR1-81XL cut with the same restriction endonucleases (see above).
- the ligation mixture was transformed into E. coli by electroporation. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing.
- PfakDewA / pJR1-81XL Such a construct was called PfakDewA / pJR1-81XL.
- the P-factor preprotein including the first amino terminal pheromone and the fused sequence encoding the hydrophobin are under the control of the nmt81 promoter.
- Example 2a The experiment was carried out in analogy to Example 2a, but using the expression vector pJR1-81XL.
- the amplified sequences were cloned into pJR1 -81XL analogously to Example 2a.
- the amplified and cut with the restriction endonucleases Xho ⁇ and ⁇ / col DNA was cloned into the Xho ⁇ and ⁇ / col sites of the expression vector pJR1-81XL.
- Example 5 Microscopic detection of the adsorption of expressed hydrophobin on Teflon
- a fluorescence-labeled HA antibody (Molecular Probes, Cat. No. A-21287) is used for the microscopic detection of the adsorption of expressed hydrophobin on Teflon.
- Transformed host cells produced according to one of Examples 1 to 4, are cultivated. Cells and any supernatant are harvested separately. Cells which have been transformed and cultured with a corresponding vector without hydrophobic genes or corresponding culture supernatants serve as reference sample.
- Teflon plates are incubated at room temperature for 18 h in cell disruption or supernatant, rinsed with water (3 x 10 min).
- the treated teflon is then incubated in PBS with fluorescence-labeled antibody.
- PBS 3 x 15 min
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JP2006525798A JP2007505602A (ja) | 2003-09-16 | 2004-09-15 | 酵母からのタンパク質の分泌 |
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US20210363199A1 (en) * | 2020-05-20 | 2021-11-25 | Industry Foundation Of Chonnam National University | Method for soluble expression and purification of hydrophobin |
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EP1848734A2 (de) * | 2005-02-07 | 2007-10-31 | Basf Aktiengesellschaft | Verfahren zum beschichten von oberflächen mit hydrophobinen |
CA2610785A1 (en) * | 2005-06-06 | 2006-12-14 | Basf Aktiengesellschaft | Method for coating fibre substrate surfaces |
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US20110126143A1 (en) * | 2009-11-24 | 2011-05-26 | Williams Michael D | System and method for interfacing fitness equipment and physiological monitoring |
EP2631296A1 (de) | 2012-02-22 | 2013-08-28 | Kimmo Koivu | Verfahren zur Hydrophobinherstellung in Pflanzen und Verfahren zur Herstellung von Hydrophobinmultimeren in Pflanzen und Mikroben |
WO2021112540A1 (ko) * | 2019-12-02 | 2021-06-10 | 주식회사 엘지화학 | Cho 세포 유래 단백질 분비 인자 및 이를 포함하는 발현 벡터 |
CN115701464B (zh) * | 2021-08-02 | 2024-06-11 | 江苏金太阳纺织科技股份有限公司 | 超疏水整理剂及其制备方法与应用 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0252561A2 (de) * | 1986-07-11 | 1988-01-13 | SCLAVO S.p.A. | Expressions- und Sekretionsvektor in Hefe, verwendbar zur Herstellung von heterologen Proteinen |
EP0662515A1 (de) * | 1993-12-10 | 1995-07-12 | Korea Institute Of Science And Technology | Signalsequenzen für die Secretion heterologer Proteine von Hefe |
WO1996041882A1 (en) * | 1995-06-12 | 1996-12-27 | Proefstation Voor De Champignoncultuur | Hydrophobins from edible fungi, genes, nucleotide sequences and dna-fragments encoding for said hydrophobins, and expression thereof |
EP0773296A1 (de) * | 1995-02-03 | 1997-05-14 | Asahi Glass Company Ltd. | Gen für ein sekretionssignal und dieses enthaltender vektor |
WO2002020651A2 (en) * | 2000-09-06 | 2002-03-14 | Zymogenetics, Inc. | Human phermone polypeptide |
WO2002046369A2 (en) * | 2000-12-08 | 2002-06-13 | Septegen Limited | Yeast-based assays involving gpcrs |
EP1223219A2 (de) * | 1997-10-31 | 2002-07-17 | Asahi Glass Company Ltd. | Induzierbarer Promoter und Sekretionssignal zur Verwendung in Schizosaccharomyces pombe, Expressionsvektor diese enthaltend und ihre Verwendungen |
WO2003053383A2 (fr) * | 2001-12-14 | 2003-07-03 | L'oreal | Utilisation cosmetique d’au moins une hydrophobine pour le traitement des matieres keratiniques |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5010182A (en) * | 1987-07-28 | 1991-04-23 | Chiron Corporation | DNA constructs containing a Kluyveromyces alpha factor leader sequence for directing secretion of heterologous polypeptides |
JP3508157B2 (ja) * | 1993-05-19 | 2004-03-22 | 旭硝子株式会社 | 分裂酵母接合フェロモン前駆体遺伝子 |
GB2281421B (en) * | 1993-08-23 | 1998-04-01 | Advanced Risc Mach Ltd | Integrated circuit |
JP4341859B2 (ja) * | 1996-12-13 | 2009-10-14 | ノバルティス バクシンズ アンド ダイアグノスティックス, インコーポレーテッド | 酵母での異種タンパク質の発現の方法 |
-
2003
- 2003-09-16 DE DE10342794A patent/DE10342794A1/de not_active Withdrawn
-
2004
- 2004-09-15 WO PCT/EP2004/010346 patent/WO2005033316A2/de not_active Application Discontinuation
- 2004-09-15 EP EP04765253A patent/EP1664306A2/de not_active Withdrawn
- 2004-09-15 CA CA002537492A patent/CA2537492A1/en not_active Abandoned
- 2004-09-15 US US10/572,189 patent/US20070077619A1/en not_active Abandoned
- 2004-09-15 JP JP2006525798A patent/JP2007505602A/ja not_active Withdrawn
- 2004-09-15 CN CNA2004800265223A patent/CN1852983A/zh active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0252561A2 (de) * | 1986-07-11 | 1988-01-13 | SCLAVO S.p.A. | Expressions- und Sekretionsvektor in Hefe, verwendbar zur Herstellung von heterologen Proteinen |
EP0662515A1 (de) * | 1993-12-10 | 1995-07-12 | Korea Institute Of Science And Technology | Signalsequenzen für die Secretion heterologer Proteine von Hefe |
EP0773296A1 (de) * | 1995-02-03 | 1997-05-14 | Asahi Glass Company Ltd. | Gen für ein sekretionssignal und dieses enthaltender vektor |
WO1996041882A1 (en) * | 1995-06-12 | 1996-12-27 | Proefstation Voor De Champignoncultuur | Hydrophobins from edible fungi, genes, nucleotide sequences and dna-fragments encoding for said hydrophobins, and expression thereof |
EP1223219A2 (de) * | 1997-10-31 | 2002-07-17 | Asahi Glass Company Ltd. | Induzierbarer Promoter und Sekretionssignal zur Verwendung in Schizosaccharomyces pombe, Expressionsvektor diese enthaltend und ihre Verwendungen |
WO2002020651A2 (en) * | 2000-09-06 | 2002-03-14 | Zymogenetics, Inc. | Human phermone polypeptide |
US20030049726A1 (en) * | 2000-09-06 | 2003-03-13 | Holloway James L. | Human phermone polypeptide |
WO2002046369A2 (en) * | 2000-12-08 | 2002-06-13 | Septegen Limited | Yeast-based assays involving gpcrs |
WO2003053383A2 (fr) * | 2001-12-14 | 2003-07-03 | L'oreal | Utilisation cosmetique d’au moins une hydrophobine pour le traitement des matieres keratiniques |
Non-Patent Citations (3)
Title |
---|
DATABASE WPI Section Ch, Week 199507 Derwent Publications Ltd., London, GB; Class B04, AN 1995-047902 XP002319523 -& JP 06 327481 A (ASAHI GLASS CO LTD) 29. November 1994 (1994-11-29) * |
IMAI Y ET AL: "THE FISSION YEAST MATING PHEROMONE P-FACTOR: ITS MOLECULAR STRUCTURE, GENE STRUCTURE, AND ABILITY TO INDUCE GENE EXPRESSION AND G1 ARREST IN THE MATING PARTNER" GENES AND DEVELOPMENT, COLD SPRING HARBOR, NY, US, Bd. 8, Nr. 3, 1994, Seiten 328-338, XP001120726 ISSN: 0890-9369 * |
NAKARI-SETALA TIINA ET AL: "Expression of a fungal hydrophobin in the Saccharomyces cerevisiae cell wall: Effect on cell surface properties and immobilization" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Bd. 68, Nr. 7, Juli 2002 (2002-07), Seiten 3385-3391, XP002319522 ISSN: 0099-2240 * |
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JP2008545435A (ja) * | 2005-06-10 | 2008-12-18 | ビーエーエスエフ ソシエタス・ヨーロピア | 新規システイン欠乏ハイドロフォビン融合タンパク質、その製造およびその使用 |
US7910699B2 (en) | 2005-06-10 | 2011-03-22 | Basf Se | Cysteine-depleted hydrophobin fusion proteins, their production and use thereof |
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WO2007014897A1 (de) * | 2005-08-01 | 2007-02-08 | Basf Se | Verwendung von grenzflächenaktiven, nicht-enzymatischen proteinen für die textilwäsche |
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US8096484B2 (en) | 2006-08-15 | 2012-01-17 | Basf Se | Method for the production of dry free-flowing hydrophobin preparations |
US8455107B2 (en) | 2007-03-12 | 2013-06-04 | Basf Se | Method of treating cellulosic materials with hydrophobins |
WO2008110456A3 (en) * | 2007-03-12 | 2009-05-22 | Ciba Holding Inc | Method of treating cellulosic materials with hydrophobins |
WO2008110456A2 (en) * | 2007-03-12 | 2008-09-18 | Basf Se | Method of treating cellulosic materials with hydrophobins |
WO2012004255A1 (de) * | 2010-07-07 | 2012-01-12 | Basf Se | Zusammensetzung enthaltend ein hydrophobin und verfahren zum reinigen von hydrophoben oberflächen |
WO2020188015A1 (en) | 2019-03-21 | 2020-09-24 | Onxeo | A dbait molecule in combination with kinase inhibitor for the treatment of cancer |
WO2021089791A1 (en) | 2019-11-08 | 2021-05-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the treatment of cancers that have acquired resistance to kinase inhibitors |
WO2021148581A1 (en) | 2020-01-22 | 2021-07-29 | Onxeo | Novel dbait molecule and its use |
US20210363199A1 (en) * | 2020-05-20 | 2021-11-25 | Industry Foundation Of Chonnam National University | Method for soluble expression and purification of hydrophobin |
Also Published As
Publication number | Publication date |
---|---|
CA2537492A1 (en) | 2005-04-14 |
DE10342794A1 (de) | 2005-04-21 |
WO2005033316A3 (de) | 2005-10-06 |
EP1664306A2 (de) | 2006-06-07 |
JP2007505602A (ja) | 2007-03-15 |
US20070077619A1 (en) | 2007-04-05 |
CN1852983A (zh) | 2006-10-25 |
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