WO2001088163A1

WO2001088163A1 - Method for producing recombinant expansins

Info

Publication number: WO2001088163A1
Application number: PCT/EP2001/005078
Authority: WO
Inventors: Frank Berendes; Hans-Georg Rast; Uwe Vogt; Christos Gouloudis
Original assignee: Bayer Aktiengesellschaft
Priority date: 2000-05-16
Filing date: 2001-05-04
Publication date: 2001-11-22
Also published as: WO2001088163A9; AU2001263895A1

Abstract

The invention relates to a method for producing recombinant expansins in micro-organisms, to the required vectors, to host cells containing these vectors, as well as to the use of these recombinant expansins.

Description

Process for the production of recombinant expansins

The invention relates to a process for the production of recombinant expansins in microorganisms, the vectors required therefor, host cells containing them

Vectors, as well as the use of recombinant expansins.

Expansins are proteins that occur in plant cell walls and are able to catalyze the linear expansion of cell walls under tension without hydrolyzing the polysaccharides contained in the cell walls

(McQueen-Mason et al., PNAS USA 91, 1994, 6574-6578).

The special property of the expansins, the structure and thus the physical properties of polysaccharides, in particular of cellulose and hemicellulose, to change makes these proteins interesting for a variety of applications in the fiber processing industry. In order to use expansins for such applications, it is necessary to have them available in sufficient quantities.

The isolation of expansins from vegetable raw material is described in US Pat. No. 5,959,082. A disadvantage of this process is that the expansins can only be isolated in small amounts. The method also includes a large number of work and cleaning steps. The process is therefore uneconomical for large-scale use.

The above problems can arise with the production of recombinant

Expansins are solved. Previous attempts to express active expansins in microorganisms such as bacteria or fungi have failed (Cosgrove et al., PNAS USA 94, 1997, 6559-6564; Grobe et al., Eur. J. Biochem. 263, 1999, 33-40 ). The object of the present invention was to provide a method which allows the production of active recombinant expansins in microorganisms.

Surprisingly, a process for the production of expansins has now been found, which is characterized in that it

a) cultivating a host cell containing a nucleic acid coding for expansins or a vector comprising a nucleic acid coding for expansins and a functionally linked sequence which ensures the expression of the nucleic acid in the host cell under conditions which ensure this expression and

b) the extraction of the expansins from the cell or the culture medium

includes.

According to the present invention, the term expansins is understood to mean all proteins which show structure-changing activity towards polysaccharides without hydrolyzing them. They consist of a polypeptide chain with a molecular weight of 25 to 30 kDa, which has more than 60% sequence similarity at the amino acid level to the S1 expansine of the cucumber Cucumis sativus (SEQ ID NO: 1).

Nucleic acids coding for expansins are all those coding for proteins, which show structure-changing activity towards polysaccharides without hydrolyzing them and consist of a polypeptide chain with a molecular weight of 25 to 30 kDa, which are more than 60% sequence similarity at the amino acid level to the S1 expansine the cucumber has Cucumis sativus (SEQ JD NO: 1). Preferably, nucleic acids that code for expansins are those that code for a protein of the amino acid sequence according to SEQ LD NO: 1 -4. The expansins produced by the process according to the invention can be in the form of "mature" proteins or as parts of larger proteins, for example as fusion proteins. Furthermore, they can have secretion or “leader” sequences, sequences that allow easy purification, such as multiple histidine residues or additional stabilizing amino acids, as long as they still show structure-changing activity against polysaccharides.

The proteins produced by the method according to the invention can also only be fragments of expansins, as long as they have a structure-changing activity towards polysaccharides.

The expansins produced by the process according to the invention can have deletions or amino acid substitutions compared to naturally occurring expansins, as long as they have at least one structure-changing activity against polysaccharides. Conservative substitutions are preferred.

Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:

1. small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. polar, positively charged residues: His, Arg and Lys;

4. large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Tip.

The following list shows preferred conservative substitutions:

The present invention also relates to vectors containing a nucleic acid coding for expansins and for proteins which have a sequence which is functionally linked to the nucleic acid and which enables the expression of the nucleic acid in microorganisms. Nucleic acids coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4 are preferably used. The nucleic acids encoding expansins (hereinafter referred to as nucleic acids) contained in the vectors according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). Preferred embodiments are fragments of genomic DNA, which may contain introns, and cDNAs. A particularly preferred embodiment is cDNA.

The sequences linked functionally to the nucleic acid are preferably regulatory sequences such as, for example, at least one promoter for constitutive or inducible expression or enhancers.

Vectors which ensure expression of the nucleic acid in bacteria and fungi are preferred.

Vectors which express the nucleic acid in. Are particularly preferred

Bacteria, preferably Gram-negative bacteria.

Vectors which enable expression of the nucleic acid in E. coli are very particularly preferred. Suitable promoters for the expression of the expansins which can be prepared by the process according to the invention in E. coli include natural hybrid or bacteriophage promoters. These are preferably promoters from the group of the λ promoters, omp, lac, trp or synthetic promoters (see also WO 98/15625, DE-A-3 430 683, EP-A-173 149) , Suitable vectors are commercially available, for example the vectors of the pET series (for example pET3a, pET23a, pET28a with His tag or pET32a with His tag) or pGEX with glutathione synthetase fusion.

The vectors pProExHT (Gibco BRL) or pASK-IBA3 (Institute for Bioanalytics, Göttingen) are very particularly preferably used. These are vectors which allow inducible expression of the expansins in E. coli, the recombinant expansins at the N-terminus having a by pProExHT 6-histidine tag are fused and fused to a streptavidin tag by pASK-EBA3 at the C-terminus.

The vectors which enable expression of the nucleic acid in E. coli can then be transformed, for example, into DE3-lysogenic E. coli strains, for example BL21 (DΕ3), HM S 174 (DE3) or AD494 (DE3). They are preferably transformed into BL21 (DE3). The vectors pProExHT and pASK-IBA3, which enable expression of the nucleic acid, are very particularly preferably transformed into BL21 (DE3).

The present invention further relates to host cells of microorganisms containing a nucleic acid coding for expansins. Host cells of microorganisms are preferred, containing a nucleic acid coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4.

The nucleic acid contained in the host cell coding for expansins, preferably coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4 (hereinafter referred to as nucleic acid) can be inserted into the genome of the host cell or can be present in a vector. The nucleic acid is preferably present in a vector comprising this nucleic acid and a sequence which is functionally linked therewith and which enables the expression of the nucleic acid in the host cell.

The host cells are preferably a fungal or bacterial cell, such as cells of Gram-negative bacteria, Gram-positive bacteria, yeast or filamentous fungi.

Preferred examples of bacteria are Streptomyces sp., Bαcillus subtilis, Sαlmonellα typhimurium, Serrαtiα mαrcescens or Pseudomonαs species. E. coli is particularly preferred. DE3-lysogenic E. coli strains are very particularly preferred, for example BL21 (DE3), HM S 174 (DE3) or AD494 (DE3). Preferred examples of yeasts are Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe.

Preferred examples of filamentous fungi are Aspergillus species.

In the process according to the invention for producing expansins, the nucleic acid coding for expansins or a vector containing this nucleic acid can be introduced into the suitable host cell using methods which are known to the person skilled in the art.

The transformed host cells of microorganisms can be cultivated in complexes or mineral media at the temperatures suitable for the particular microorganism. The respective microorganisms are preferably grown to high cell densities. The transformed microorganisms are particularly preferably cultivated up to the late exponential / early stationary phase.

If vectors are used in the method according to the invention which require induction of the expansin synthesis, the induction is preferably carried out at temperatures between 1 and 30 ° C., particularly preferably between 4 and 28 ° C., very particularly preferably between 10 and 20 ° C.

In a preferred embodiment of the method according to the invention, the transformed microorganisms are cultivated up to the late exponential / early stationary phase and the induction of the expansin synthesis is preferably carried out at temperatures between 1 and 30 ° C.

The induction of the expansin synthesis is preferably carried out for a period of from 10 minutes to 48 hours, particularly preferably from 20 minutes to 16 hours, very particularly preferably from 30 minutes to 6 hours. In a very particularly preferred embodiment of the method according to the invention, the cultivation of the transformed microorganisms up to the late exponential / early stationary phase and the induction of the expansin synthesis preferably take place at temperatures between 1 and 30 ° C. for a period of 10 minutes to 48 hours.

The host cells are preferably disrupted at temperatures from 0 to 30 ° C., preferably at 1 to 25 ° C., particularly preferably at 4 to 20 ° C. To protect the recombinant expansins from proteolytic cleavage, a protease inhibitor is preferably added.

A method for producing expansins, which is characterized in that

a) the cultivation of E. coli cells containing a vector comprising a nucleic acid coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4 as well as a regulatory sequence associated therewith which ensures the expression of the nucleic acid in E. coli, occurs until the late exponential early stationary phase,

b) the induction of the expansin synthesis takes place at temperatures of 1 to 30 ° C in a period of 10 minutes to 48 hours and

c) the extraction of the expansins by disruption of the cells

Temperatures from 0 to 30 ° C, preferably at 1 to 25 ° C, particularly preferably at 4 to 20 ° C.

To protect the recombinant expansins from proteolytic cleavage, a protease inhibitor is preferably added when the expansins are obtained by disrupting the cells. The analysis of the expansins obtained by the method according to the invention can be carried out, for example, by Western emblot. The recombinant expansins can be detected, for example, via fused tags.

In order to check the solubility of the expansins obtained by the process according to the invention, the crude extracts or supernatants, which can be obtained by centrifuging the crude extracts, can be separated electrophoretically in SDS-PAGE.

If the expansins obtained in the process according to the invention are fusion proteins, they can be affinity-purified, for example.

In the expansins obtained in the process according to the invention, the activity can be detected by all methods and techniques with which a structure-changing effect on polysaccharides can be measured. Particularly suitable are methods for determining the strength of paper, cotton thread or other cellulose-containing or hemicellulose-containing materials, or methods for determining the mechanical properties required for tearing paper, cotton fibers or other cellulose-containing or hemicellulose-containing materials

Energy. The methods described in McQueen-Mason and Cosgrove, PNAS USA 91, 1994, 6574-6578 and McQueen-Mason et al, Planta 4, 1992, 1425-1433 are also suitable for detecting the activity.

For a better understanding, the meaning of certain words and terms used in the description, examples and appended claims will be explained in more detail below.

The term "fragments" in relation to proteins and DNA describes parts of expansins or parts of those under SEQ ID NO. 1-4 described nucleic acid or amino acid sequence which have at least one structure-changing activity towards polysaccharides.

The term "vector" describes a DNA element that is used to introduce exogenous nucleic acids into host cells. A vector contains a nucleic acid sequence that codes for one or more polypeptides. Vectors that are capable of directing the expression of the genes that contain them are also referred to as "expression vectors".

The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acids (DNA) or, if appropriate, to ribonucleic acids (RNA). The term also includes, in an equivalent manner, analogs of RNA or DNA which are produced from nucleotide analogs, and, if appropriate, single-stranded (“sense” or “antisense”) and double-stranded polynucleotides.

The term "promoter" refers to DNA sequences that regulate the expression of a particular DNA that are functionally linked to the promoter. The term also includes "tissue-specific" promoters, i.e. Promoters that control the expression of the specific DNA only in certain cells (for example cells of a certain tissue). Also included are "non-tissue-specific"

Promoters and promoters that lead to constitutive expression or are inducible.

A “fusion protein” is a fusion of a first amino acid sequence coding for one of the above-described expansins with a second amino acid sequence which has no commonality or basic homology to the expansin sequences. The second amino acid sequence can originate from the same organism as the first, or alternatively originate from another organism (intergenic). In general, a fusion protein can be represented using the formula X-Expansin-Y. X and Y stand for a polypeptide that is not linked together. with an expansin amino acid sequence. X or Y can each be absent independently.

The terms “cell” or “host cell” can be used in the same sense in the context of the present disclosure. It is understood that these terms refer not only to a single cell, but also to the descendants of such a cell. Due to certain modifications in the course of subsequent generations (e.g. mutations), such offspring may not be identical to the stem cell, but are included in the present invention.

The term "Tntron" describes such sequences of the described, preferably genomic DNA, which are transcribed, but then removed from the transcript by so-called "splicing", the adjacent sequences (exons) being linked.

nucleic acids

Nucleic acids coding for expansins, as described above, can be obtained starting from mRNA which is present in plant cells. It is also possible to obtain the DNA according to the invention from genomic DNA from the relevant plant cells. A gene coding for an expansin can be obtained, for example, from a cDNA or a genomic DNA library. cDNA can be obtained by isolating the entire mRNA of a cell. Starting from the mRNA, double-stranded cDNA can then be produced and inserted into a suitable expression construct or a suitable vector. The DNA necessary for the method according to the invention can also be obtained by amplification using the known polymerase chain reaction (PCR) or else by de novo DNA synthesis (see also J. Sambrook et al. (1989) Molecular Cloning, 2. Edition, chap. 14). vectors

The present invention also includes vectors which contain a nucleic acid coding for expansins, preferably coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4, which are functionally linked to a regulatory sequence. "Functions linked" means that the nucleic acid sequence is linked to the regulatory sequence in such a way that the expression of the protein encoded by the nucleic acid sequence can be controlled. "Regulatory sequences" include, for example, promoters, enhancers and other control elements. The vectors contain, for example, a gene coding for an expansin or fragments thereof. These vectors can be used to be introduced into cells, where the corresponding expansins or fusion proteins thereof then arise. The transformation can be carried out using methods known to those skilled in the art, such as ion-mediated transformation, electrotransformation or transfection.

Expression of the expansins

The present invention also encompasses host cells of microorganisms which have a nucleic acid coding for expansins, preferably coding for a protein of the

Amino acid sequence according to SEQ LD NO: 1-4, contained (e.g. in a vector, an expression construct or inserted into the genome). Suitable microorganisms are, for example, bacteria, yeast or filamentous fungi.

The basic steps to make recombinant expansins are:

1. Obtaining a natural, synthetic or semi-synthetic DNA which codes for expansins in the sense of the invention. 2. Introduction of this DNA into an expression construct or a vector which is suitable for expressing expansins, either alone or as fusion proteins.

3. Transformation of a suitable host cell of a microorganism with this expression construct or vector.

4. Cultivate this transformed host cell in a manner suitable for expressing expansins.

5. Purification of the expansins obtained from the cells or from the culture medium by suitable, known methods.

For example, vectors containing a nucleic acid encoding expansins in λDE3-lysogenic E. co / z strains, e.g. BL21 (DE3), HM S174 (DE3) or

AD494 (DE3) can be transformed. After the cells have grown, expression is induced with IPTG. After a incubation period, the cells are disrupted, the expressed protein purified by chromatographic methods, in the case of protein expressed with His tag by FPLC on a Ni-NTA column, in the case of protein expressed with streptavidin tag on a biotin column, as well as by ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or else immunoaffinity purification, which are specific for the expansins.

Homologs or fragments of those produced in the process according to the invention

Expansins can be generated by mutagenesis, such as directed (point) mutagenesis, or by deletions.

When expressing the expansins according to the present invention, it may be advantageous to change certain codons in order to enable optimal expression. This applies if the use of certain codons ("Codon usage ") is different in the heterologous expression system than in plants. Furthermore, the deletion of the 5'- or 3'-untranslated region is possible, for example if there are several destabilizing sequence motifs (eg ATTTA) in the 3 'region of the cDNA.

fusion proteins

The expansins produced in the process according to the invention can also be present as part of a fusion protein. Such fusion proteins are fully encompassed by the present invention. Fusion proteins facilitate expression of a protein under certain circumstances. For example, the expansins can be made as glutathione-S-transferase (GST) fusion proteins. Such GST fusion proteins allow easy protein purification (see e.g. Current Protocols in Molecular Biology, eds. Ausubel et al. (John Wiley & Sons, N.Y. 1991). Fusion proteins can contain, for example, a "leader" sequence that corresponds to the

Purification serves, for example, a poly-histidine sequence at the N-terminus (but also at the C-terminus) of the protein allows it to be purified by chromatography on a Ni ²⁺ NTA column (see, for example, Hachuli et al. (1987) J. Chromatography 411, 177).

Techniques for producing such fusion proteins are known to the person skilled in the art.

The recombinant expansins which can be prepared by the process according to the invention can be used in the recycling of paper due to their structure-changing activity towards polysaccharides, in particular towards cellulose and hemicellulose. In addition, they can be used to produce pulp from vegetable tissue and thus to substitute aggressive chemicals in paper production.

The recombinant expansins which can be prepared by the process according to the invention can, owing to their stoic-altering activity towards polysaccharides, in particular with respect to cellulose and hemicellulose, also in the production, treatment and finishing of textiles based on cellulose, such as cotton.

Examples

example 1

PCR cloning of expansin genes

All expansenes were amplified using the PCR method either from self-made or from commercially acquired cDNA banks. The published original sequences, the primers used for the amplification and the amplified expandans genes are listed in Table 1.

Table 1

1.1 Creation of a cDNA bank from cucumber seedlings

Seeds of the cucumber variety Cucumis sativus variety euphya (Enza Zaden, the Netherlands) were incubated in glass dishes on moist filter paper at 25 ° C in the dark. After the roots of the seedlings had reached a length of between 2 and 6 cm, they were separated, frozen in liquid nitrogen and stored at -80 ° C. The entire RNA was isolated from 3 g of the frozen seedling roots using the phenol / SDS method for isolating plant RNA (Ausubel et al., 1994).

The poly (A) ⁺ RNA was isolated from the total RNA using the “mRNA direct kit” (Dynal, Hamburg). Using the "superscript preamplification

Systems "(Gibco BRL Life Technologies, Heidelberg), the first strand synthesis of the cDNA was carried out on the basis of the poly (A) ⁺ RNA bound to magnetic" beads ", also according to the manufacturer's instructions.

1.2 Amplification of the Expansingen

10 ⁶ pfu (plaques forming units) to 5 μl high-fidelity buffer (Gibco BRL Life Technologies, Heidelberg) were pipetted from the commercially acquired cDNA banks and incubated for 30 min at 70 ° C. to the c-DNA packaged in phages to be released as a template for the PCR. In the case of the cucumber cDNA bank, 80 μg of the cDNA bound to the beads were treated with 5 μl of high-fidelity ^' P-Ser (Gibco BRL

LifeTechnologies, Heidelberg) mixed and used as a template without prior incubation at 70 ° C.

The mixtures were 0.2 mM dNTPs (Clontech Laboratories GmbH, Heidelberg), 1 mM MgSO ₄ , 0.2 uM primer forw / rev (sequence see below), another 0.5 ul high

Fidelity volume enzyme (Gibco BRL LifeTechnologies, Heidelberg) and H ₂ O αd 50 μl added. In a T3 thermal cycler from Biometra, Göttingen, the amplification was carried out according to the following temperature program:

Primers used

No.2: forward primer csexpOl

5 '-ttcttctttgtcttcaccgaattcgactacggtggctggcag-3'

No.3: reverse primer csexpOl

5 '-gcagtgtggctgatgcggccgcgaattgagggccttcatagg-3'

No.4: forward primer leexp03 5 '-ttactaacactcacactggaattcacattctcactagctcac-3'

No.5: reverse primer leexp03

5 '-tcaaggaaggtccagagcggccgccgattcgaactgcttacc-3'

No.6: forward primer ntexp04 5 '-gcaatggtctcatcagttgaattctatggtggaggaggttgg-3'

No.7: reverse primer ntexp04

5 '-atattaaatgcttgagcaggcggccgcctgcaggtggaattgagctccagtata-3'

The PCR products were analyzed using the "TOPO TA Cloning Kit" (frivitrogen,

Leek, Netherlands) was cloned into the pCR2.1 -TOPO vector and electrotransformed into competent E. coli DH12S cells (Gibco BRL LifeTechnologies, Heidelberg). The hybrid plasmids were then isolated from the corresponding E. coli DH12S clones using the “QIAprep Spin Miniprep Kit” (Qiagen, Hilden).

The expansion genes inserted in the pCR2.1-TOPO vector were sequenced using the primers recommended by the manufacturer and compared with the literature data. The sequence analysis revealed the identical sequence for the tomato expansin (leexp3, SEQ ID NO: 3) and for the cucumber expansin (csexpla, SEQ ID NO: 2) a sequence which had three silent mutations but no change in the amino acid sequence compared to the published sequence , In the case of Täbakexpansins (ntexp4a, SEQ ID NO: 4) a gene was identified which has 14 mutations and 2 amino acid changes compared to the published sequence.

Example 2

Cloning of the expansion genes in expression vectors

For transfer into the expression vectors, the amplified expansion genes were cut out from the pCR2.1-TOPO vectors by restriction digestion, separated into agarose gel and isolated from the agarose gel with the aid of the “QIAEX II Agarose Gel Extraction Kit” (Qiagen, Hilden). In another Ligation reaction, they were inserted as EcoRI-Nσtl fragments in the pProΕxHTaS vector (Gibco LifeTechnologies, self-modified) and as EcoRMTzoI fragments in the pASK-IB A3 vector (Institute for Bioanalytics, Göttingen). Since the target vector pASK-IBA3 none Contains the Not / interface, the Εxpansine genes were cloned from the pProΕxHTaS vector as EcoRI-Notl fragments, cloned into the pProΕXHtΕXb vector (Gibco BRL LifeTechnologies) and as EcoRI- _^ ÖzoI before they were cloned into the pASK-LB A3 vector The pProΕxHTaS vector differs from the pProΕxHTa vector in that from position 396 to 401 there are two additional stop codons The original sequence -tcgaat- was changed to -gactga-.

All expression vectors were transformed into competent E. coli BL21 (DΕ3) cells (Stratagene, La Jolla, CA, USA). By cloning in the pProExHT

Vector, the recombinant proteins at the Ν-terminus were fused with a 6-histidine tag and by cloning into the pASK-IB A3 vector at the C-terminus with a streptavidin tag (see scripts of the manufacturers). Example 3

Expression of the expansins in E. coli

To express the expansins, the recombinant E. coli strains were

Medium with 100 μg / ml ampicillin at 37 ° C. The growth of the cultures was _monitored by determining the optical density at a wavelength of 600 nm (OD _ÖOO ). After an OD ₆₀₀ of 5 had been reached, the induction of the expansin synthesis was carried out by adding 1 mM LPTG (pProExHT vector) or 2 mg / 1 anhydrotetracycline (pASK-LBA3 vector). The expression cultures were incubated for 4 h at 20 ° C. and then sedimented by centrifugation at 6000 × g and 4 ° C. for 15 min. After the pellet had been taken up in 1/100 volume of lysis buffer (100 mM TrisHCl pH 8, 1 mM EDTA), the cells were disrupted by ultrasound treatment at 4 ° C. To protect the proteolytic cleavage of the recombined expansins, 0.4 mg / ml of the protease inhibitor was added to the lysis buffer

Pefabloc SC (from Boehringer, Mannheim) added.

In order to check the solubility of the recombinant proteins, the crude extracts and their clear supernatants were separated electrophoretically in SDS-PAGE. The clear supernatants were prepared by centrifuging the crude extracts for 30 min at 16,000 x g and 4 ° C.

The recombinant expansins were detected in the Westerblot via their fusion components. The 6-His-Tag-coupled proteins were detected using a monoclonal ANTI-polyHISTIDINE Clone His-1 antibody

(Sigma Chemical Co., St. Louis, USA) and an ANTI-MOUSE IgG antibody coupled with an alkaline phosphatase (Sigma Chemical Co., St. Louis, USA). The strep-tag-coupled proteins were detected using a "streptavidin alkaline phosphatase conjugate" (Institute for Bioanalytics, Göttingen). The detection was carried out according to the manufacturer's instructions. Under the conditions described, all three genes could be expressed to soluble recombinant proteins, which could be identified in the western emblot on the basis of clear signals at the level of the expected molecular weights (CsexpOla: 27.5 kDa; Leexp03: 30.5 kDA, Ntexo04a: 27.5 kDA ).

Example 4

Change in the strength of paper by recombinant tomato expansin

The gene of tomato expansin cloned into the pASK-IBA 3 expression vector was, as described in Example 3, expressed in E. coli BL21 (DE3). The cells were sedimented and resuspended in 1/100 vol. Lysis buffer.

After ultrasonic lysis, the insoluble components of the crude extract were removed

Centrifugation (16,000 xg, 30 min, 4 ° C) separated and the soluble supernatant with a cellulose ester membrane (Spectra / Por CE, MWCO: 10,000, Spectrum Medical Industries Inc., Houston, Texas, USA) for 4 h at 4 ° C dialyzed against 50 mM sodium acetate buffer pH 4.5.

Four 1.5 x 4 cm paper strips (Whatman No. 3, Whatman International Ltd., Maidstone, England) were each stretched between two stainless steel clamps, weighted with a 100 g weight and in pH with 50 mM sodium acetate buffer 4.5 filled 50 ml measuring cylinders hung. After the strips had hung for 2 hours, 5 ml of the dialyzed soluble crude extract were added in two batches. As a control, the same amount of sodium acetate buffer pH 4.5 was added to the other two batches. The influence of the addition of protein on the strength of the paper strips was observed for 3 hours. In the two batches of dialyzed soluble crude extract, the paper strips tore off after 58 and 65 minutes respectively, while in the control batches they remained stuck for longer than 3 hours. The use of soluble raw extracts from cultures in which for The vector pProExHt-CAT (Gibco BRL LifeTechnologies, Heidelberg) had a chloramphenicol acetyl transferase (CAT) expressed (control experiment), also showed no reduction in paper strength.

Example 5

Affinity purification of the recombinant tomato expansin

The strain E. coli BL21 (DE3) with the expression vector pASK-IBA3: leexp3 was grown at 37 ° C. in 2.4 liter LB medium in the presence of 100 μg / ml ampicillin. After an OD ₆₀₀ of 5 had been reached, the incubation temperature was lowered to 20 ° C. and the synthesis of the recombinant tomato expansin was induced by adding 2 mg / 1 anhydrotetracycline (AHT). After a further 4 h, the cells were sedimented by centrifugation, resuspended in 1/100 volume of lysis buffer and disrupted by means of ultrasound treatment.

Insoluble components were separated by centrifugation for 30 min at 4 ° C and 16,000 x g. The recombined protein was purified from the soluble supernatant of the crude extract by means of affinity chromatography. That was what

"Strep-Tag Ü-System" (Institute for Bioanalytics, Göttingen) was used. The implementation was carried out according to the manufacturer's protocol. The protein content of the individual fractions was measured with the BIO-RAD PROTEIN ASSAY, (BIO-RAD Laboratories, Munich).

After SDS-PAGE and Westemblot analysis, signals were identified in 4 fractions, which could be assigned to the expected molecular weight of tomato expansin of 30.5 kDa. Example 6

Influence of recombinant affinity-purified tomato expansin on the strength of paper

In order to be able to measure the influence of the recombinant tomato expansin (see Example 5) purified by affinity chromatography, the stress-strain profile of paper (Whatman No. 3, Whatman International Ltd., Maidstone, England) was recorded in accordance with the modified DIN 53455. In deviation from DIN 53455, paper soaked in liquid was used. The

Paper was punched out into strips of the "test specimen 3" format.

The stress-strain profile was recorded with a type 1455 universal testing machine (Zwick GmbH, Ulm). The paper strips were stretched over a distance of 7.5 cm at a speed of 0.5 mm / min, the applied tension σ in N / mm ² and the elongation ε being measured in% of the strip length. For the evaluation, the mechanical energy related to the area was used (ME [mm * MPa]), which had to be used to tear the paper. This size characterizes the strength of the paper and is calculated from the integral of the stress-strain function.

Four approaches, each with four strips of paper, were placed in glass dishes. Based on one strip each, the paper was moistened with the solutions listed below before the batches were filled with 80 ml of 50 mM sodium acetate buffer pH 4.5 and incubated for 60 minutes at room temperature. The stress-strain profile was then recorded. In addition to the affinity-purified tomato expansin and dissolved in elution buffer (script for the Strep-Tag II system, Institute for Bioanalytics, Göttingen), bovine serum albumin (BSA; Boehringer, Mannheim) and pure elution buffer were also used as controls. Batch 1 (according to the invention): 650 μl (0.4 ml 500 mM sodium acetate pH 4.5 + 1.0 ml

Elution buffer + recombinant, affinity-cleaned tomato expansin, total protein content 1.5 mg)

Batch 2 (control) 650 μl (0.4 ml 500 mM sodium acetate pH 4.5 + 1.0 ml elution buffer) Batch 3 (control) 650 μl (0.4 ml 500 mM sodium acetate pH 4.5 + 1.0 ml Elution buffer + bovine serum albumin, total protein content 2.0 mg))

The results are shown in Table 2

Table 2: Influence of affinity-purified recombinant tomato expansin on the strength of paper

The results of the two experiments show that recombinant tomato expansin affects the strength of the paper, whereas BSA has no significant effect even in high concentrations.

Literature:

Ausubel et al., (1987) Current Protocols in Molecular Biology, J. Wiley and Sons, New York

McQueen-Mason and Cosgrove, (1994) Proc. Natl. Acad. Be. USA, Vol 91, pp. 6574-6578, 1994

McQueen-Mason et al., (1992) Plant Cell Vol.4, pp. 1425-1433

Shcherban et al., (1995) Proc. Natl. Acad. Be. USA, Vol 92, pp. 9245-9249

Cosgrove et al., (1997) Proc. Natl. Acad. Be. USA, Vol 94, pp. 6559-6564

U.S. Patent, No. 5,959,082

McQueen-Mason and Rochange, (1999) Plant Biol., Pp. 19-25

Grobe, et al. (1999) Eur. J. Biochem. Vol. 263, pp. 33-40

Hachuli et al. (1987) J. Chromatography 411, 177

Claims

Patentansprflche

1. Vectors containing a nucleic acid coding for expansins and a functionally linked sequence which enables the expression of the nucleic acid in microorganisms.

2. Vectors according to claim 1, characterized in that the nucleic acid codes for a protein of the amino acid sequence according to SEQ ID NO: 1-4.

3. Vector according to claim 1 and 2, characterized in that with the

Nucleic acid _ unfunctionally linked sequence is a regulatory sequence.

4. Vector according to one or more of the preceding claims 1 and 3, characterized in that it enables the expression of the nucleic acid in bacteria or fungi.

5. Vector according to one or more of the preceding claims 1 and 4, characterized in that it enables expression of the nucleic acid in E. coli.

6. Vector according to claim 5, characterized in that it is pProExHT or pASK-LBA3.

7. host cell of a microorganism containing a nucleic acid coding for expansins or a vector according to claim 1.

8. Host cell according to claim 7, characterized in that it contains a nucleic acid coding for a protein of the amino acid sequence according to SEQ LD NO: 1-4 or a vector according to claim 2.

9. Host cell according to claim 7 and 8, characterized in that the microorganism is a microorganism from the series of bacteria and fungi.

10. Host cell according to one or more of the preceding claims 7 to

9, characterized in that the microorganism is E. coli.

11. Host cell according to claim 10 containing a vector from the series pProExHT and pASK-IBA3, comprising a nucleic acid coding for a

Protein of the amino acid sequence according to SEQ ID NO: 1-4 and a functionally linked regulatory sequence which enables the expression of the nucleic acid in E. coli cells.

12. Process for the production of recombinant expansins, characterized in that it

a) culturing a host cell containing a nucleic acid encoding expansins or a vector comprising a nucleic acid encoding expansins and a functionally linked thereto

Sequence which ensures the expression of the nucleic acid in the host cell under conditions which ensure this expression and

b) the extraction of the expansine from the cell or the culture medium

includes.

13. The method according to claim 12, characterized in that the nucleic acid is a nucleic acid coding for a protein of the amino acid sequence according to SEQ ID NO: 1-4.

14. The method according to claim 12 and 13, characterized in that vectors according to claims 1 to 6 are used.

15. The method according to one or more of the preceding claims 12 to

14, characterized in that the host cell is a host cell according to claims 7 to 9.

16. The method according to one or more of the preceding claims 12 to 15, characterized in that E. coli cells are used as host cells.

17. The method according to one or more of the preceding claims 12 to 16, characterized in that the cultivation of the host cells takes place up to the late exponential / early stationary phase.

18. The method according to one or more of the preceding claims 12 to 17, characterized in that an inducible vector is used and the induction of the expansin synthesis takes place at temperatures of 1 to 30 ° C.

19. The method according to claim 18, characterized in that the cultivation of the host cells takes place up to the late exponential / early stationary phase.

20. The method according to one or more of the preceding claims 18 and 19, characterized in that the induction takes place in a period of 10 minutes to 48 hours.

21. The method according to one or more of the preceding claims 12 to 20, characterized in that the recombinant expansins are obtained by disrupting the host cells.

22. The method according to claim 21, characterized in that the disruption of the host cells takes place at a temperature of 0 to 30 ° C.

23. A process for the production of recombinant expansins, characterized in that

a) the cultivation of E. coli cells containing an inducible vector comprising a nucleic acid coding for a protein of the amino acid sequence according to SΕQ ID NO: 1-4 and a regulatory sequence which is functionally linked thereto and which ensures the expression of the nucleic acid in E. coli, takes place until the late exponential / early stationary phase,

c) the expansins are obtained by disrupting the cells at temperatures from 0 to 30 ° C.

24. Recombinant expansins which can be produced by a process according to claims 12 to 23.

25. Use of recombinant expansins in the manufacture, treatment and finishing of cellulose-based textiles.