CA2047235A1 - Process for the enzymatic treatment of substrates - Google Patents

Abstract

A b s t r a c t For the enzymatic treatment of substrates, the substrate to be treated is brought into contact with a biocatalyser which was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and the enzymatically treated substrate is subsequently isolated.

Description

2 0 ~ 7 2 3 3 D e s c r i p t i o n The invention concerns a process for the enzymatic treatment of substrates.

In virtue of their substrate specificity, enzymes are extensively used in biotechnology and in diagnostics. In biotechnology enzymes are used for the specific conversion of substrates. Thus, derivatives of starch can be produced by treatment with amylase. In diagnostics specific conversions are carried out using enzymes which lead to a signal which can be evaluated.
Moreover in diagnostics it is sometimes necessary to specifically convert substrates before their detection.
Thus, for example, for the cholesterol determination cholesterol esters present in the sample must be saponified beforehand. For many of these applications it is advantageous to immobilize enzymes. As a rule, this is done by binding enzymes to a carrier material. In this process, the binding of the enzymes is usually covalent via spacers which are bound to functional groups of the enzyme. A problem in this is that the binding must be carried out in such a way that the enzymatic properties of the enzyme are not impaired by the immobilization and moreover that the binding is carried out so that the active centre remains accessible to the substrate. In addition, the binding of the enzyme should be such that no detachment occurs under the conditions during the reaction with the substrate.

Enæymes that are used for the above-mentioned purposes are often produced by means of genetic engineering. For this a gene coding for the desired enzyme is inserted into a plasmid, and transformed and expressed in a - 2 - ~V ~ 12 3 ~

suitable organism. The enzymes are isolated from the lysate after cell lysis. The purification of the enzyme often causes problems since very many proteins are present in the lysate which is obtained and for further processing it is absolutely essential that these are separated.

It was therefore the object of the invention to provide a biocatalyser for the enzymatic treatment of substrates in which the enzymes in pure form are bound in a simple manner and in such a way that their activity is not impaired and that they do not become detached under the reaction conditions during the isolation of the treated substrate and that, as a consequence thereof, the biocatalyser is available for as many conversion cycles as possible.

This object is achieved by a process for the enzymatic treatment of substrates which is characterized in that the substrate to be treated is brought into contact with a biocatalyser which was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material in order to produce a fuslon protein, thcn inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and the enzymatically treated substrate is subsequently isolated.

~0~72~

In order to produce the biocatalyser according to the present invention, the enzyme in the form of a fusion protein is immobilized by intermolecular interactions on a carrier material in a non-covalent binding in such a manner that the binding is not broken under the conditions of the substrate conversion and as a result of which the immobilized enzyme is available for further reaction cycles. Several advantages are achieved by binding via a peptide sequence fused to the N- or C-terminal end of the enzyme. The binding does not impair the active centre or the mobility~of the enzyme. In this way the accessibility of the immobilized enzyme to the substrate is improved. In addition, a purification is not necessary since the enzyme can be bound directly from the lysate to the carrier material.

Moreover, the immobilized enzyme can be detached from the carrier material in order to regenerate the biocatalyser in a manner known to the expert and the biocatalyser can be again loaded with lysate containing the enzyme without having to empty the bioreactor vessel or dispose of the carrier material.

According to the present invention a biocataly~er is used for the enzymatic treatment of substrates which contains a carrier material to which a fusion protein is bound via intermolecular interactions. This fusion protein consists of the enzyme which i5 necessary for the process and a binding peptide which mediates the binding to the carrier material. All enzymes that can be produced by genetic engineering are suitable for the process accordiny to the present invention such as e.g.
~-glucosidase, glucose oxidase, aminoacylase, glucose isomerase, creatinase, ~-galactosidase, pullulanase, trehalase, trehalose phosphorylase, glucose ~7233 dehydrogenas~, mannitol dehydrogenase, D-amino acid oxidase (D-AOD), aldolase, cholesterol esterase, alcohol dehydrogenase (ADH), pig liver esterase, subtilisin, dehalogenases, naphthaline dioxygenase, chymotrypsin, ~-amylase and thermo-stable amylases, ligninase, nitrile hydratase, horse radish peroxidase. Amino acid sequences that can be produced by genetic engineering and which can interact with a carrier material are suitable as the binding peptide. The fusion protein can be produced according to known molecular biological processes (T.
Maniatis, E.F. Fritsch and Sambrook, J. Molecular Cloning, 1982, Cold Spring Harbor Laboratory). For this, DNA fragments coding for the enzyme and the binding peptide are inserted into a suitable vector. The vector i9 then transformed in a suitable organism and, after selection, this organism is then cultured in the usual manner. The cultured cells are lysed according to known processes and the lysate obtained in the lysis which contains the fusion protein is brought into contact with the carrier material. The fusion protein binds to the carrier material via the binding peptide and can thus be separated from the other substances contained in the lysate.

A material which can interact intermolecularly with the binding peptide is used as the carrier material.
Intermolecular interactions which are suitable for immobilizing the fusion protein on the carrier material are ionic, hydrophilic interactions, complex formation, hydrophobic interactions, binding peptide/receptor interactions as well as signal peptide/membrane interactions. The carrier material and sequence of the binding peptide are therefore chosen so that they can participate in one of these interactions. Proteins and carrier materials which are suitable for this are known ~ ~ ~ 7 2 3 ~

to the expert. The binding preferably takes place via ionic interaction whereby the carrier material as well as the binding peptide of the fusion protein have charged groups or via the formation of complexes such as those used e.g. in metal-chelate affinity chromatography.

Gels such as those which are also used for chromatography or ion-exchange resins are preferably employed as the carrier materials. Carriers which can be mechanically stressed are particularly suitable, in particular hydrophilic polymerisates denoted fractogels and matrices denoted "tentacle-type" gels which carry exchanger centres on "tentacle-like" polymer chains bound to a hydrophilic matrix. Other materials are also suitable in a corresponding derivatised form such as e.g. those materials denoted soft gels which are based on polysaccharides and are well known for chromatography such as dextrins, agarose or sepharose. For example S-Sepharose~ ff~Pharmacia/LKB) CM-Sepharose~ ff (Pharmacia/LKB) SP-Sephade ~ C-50 (Pharmacia/LKB), for batch application SE-EUPERGI ~ (R~hm Pharma) Bio-Re ~ 70 (Bio-Rad) Heparin-Sepharose~ CL-6B
Fractogel~ TSK SP-650 (Toyo Soda/Merck) Fractogel~ EMD SO3 -650 (Merck, "tentacle-type gel") Q-Sepharose~ ff (Pharmacia/LKB) DEAE-Sepharose~ ff (Pharmacia/LKB) DEAE-Sephade ~ A-50 (Pharmacia/LKB), for batch application QAM-EUPERGIT~ (Rohm Pharma) AG MP-l (Bio-Rad) Fractogel~ TSK DEAE-650 (Toyo Soda/Merck) Fractogel~ EMD TMAE-650 Merck, "tentacle-type gel") - 6 - ~ 2 3 ~i can be used as derivatives. Fractogel~ EMD S03 -650 is preferably used as the carrier material when the binding peptide contains arginine and lysine as amino acids.
When glutamic acid and aspartic acid are used for the binding peptide, Fractogel~ EMD TMAE-650 is preferably used.

Chelate-forming resins such as e.g. TSK chelates 5-PW or chelating Sepharose~ 6B ff which have iminodiacetate as ligands, as well as tris-(carboxy~methyl)-ethylenediamine-agarose are suitable for binding the fusion proteins. A column material is particularly preferred as the carrier material which contains the metal chelator nitrilo-triacetic acid (NTA) as the ligand which mediates the binding to the fusion protein.
The protein sequence of the fusion protein which is responsible for the binding then contains poly-histidine. The binding then takes place via carrier-bound metal ions via formation of a complex with the functional histidine residues.

A carrier material is particularly preferably used which has negatively or positively charged groups. The protein sequence of the fusion protein which is responsible for the binding then has oppositely charged group~ which can be introduced by the amino acids lysine and/or arginine or glutamic acid and/or aspartic acid. The binding then takes place via polyionic interactions.

The binding peptide part of the fusion protein i5 composed of several amino acids, whereby the length of the peptide is chosen so that a sufficiently strong binding of the fusion protein to the carrier material is ensured and so that the active sites of the enzyme are accessible for substrate binding. The binding peptide is 3 j preferably composed of 2 to 30 amino acids. In a preferred embodiment the binding peptide is not only composed of the amino acids which have the functional groups which are necessary for the binding to the carrier material but contains in addition several other amino acids which improve the accessibility of the enzyme. The introduction of a proline or glycine polymer with 1 to 10 amino acid groups is particularly suitable.
It is expedient if the amino acid sequence of the binding protein is so chosen that it is substantially resistant to digestion by proteinases.

The binding peptide does not have to be composed of a homogeneous chain of the same amino acids. It can also have a combination of similarly charged amino acids. The binding peptide is preferably composed of a sequence which contains the amino acids arginine and/or lysine or aspartic acid and/or glutamic acid.

In order to protect the binding peptide part of the fusion protein against proteolytic digestion the free end of the binding peptide is preferably protected by an amino acid such as e.g. proline which is fused to it and that is not charged and is not easily accessible to exoproteinases.

Since the enzyme contalned in the fusion protein can be present in a partially denatured form as a result of the processing and cannot in this case develop its full activity, in a preferred embodiment the enzyme is renatured according to well-known methods after immobilizing it on a carrier in which it is first treated with a denaturing agent and subsequently a renaturation step is carried out. The conditions for this are adjusted so that no detachment of the fusion ~72~.~

protein can take place. This has the particular advantage that reaggregation, which would otherwise be a risk in the renaturation, is not possible because of the immobilization.

In order to bind the fusion protein to the carrier material, the lysate obtained after cell lysis is brought into contact with the carrier material under conditions which favour the binding. In this process the binding peptide binds via its functional groups to the corresponding functional groups of the carrier material.
The biologically active immobilized enzyme obtained in this way can then be used for the treatment of substrates. The substrate is brought into contact with the immobilized enzyme in the usual manner and subsequently the enzymatically treated substrate is isolated. The immobilized enzyme used according to the present invention is preferably filled in a column and the substrate is passed over it in a well-known manner.

A further object of the invention was to provide solid phase-bound or particle-bound, specifically bindable peptide and protein substances (receptors) for use in enzyme-immunoassays in which the receptors are bound in a pure form in a simple manner in such a way that their immunological activity is not impaired and a detachment under the reaction conditions during the immunoassay procedure does not take place.

This object is achieved by a method for the detection of specifically bindable substances according to the immunoassay principle using a solid phase-bound receptor which is characterized in that the sample solution as well as at least one labelled receptor which is capable of binding to the substance to be detected are brought ~ 3 _ g into contact with a solid phase-bound receptor which was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate which contains the fusion protein into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and the after incubation the solid phase is separated from the liquid phase and the label is determined in one of the two phases.

A method for the detection of specifically bindable substances according to the principle of the homogeneous immunoassay using a particle-bound receptor is provided as a further embodiment which is characterized in that the sample is incubated with a particle-bound receptor which is capable of binding to the substance to be detected, which receptor was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate which contains the fusion protein into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and after incubation the 3 ~

solid phase is separated from the liquid phase and the agglutination is determined after incubation.

According to the present invention the solid phase-bound or particle-bound receptor used is immobilized in the form of a fusion protein by intermolecular interactions on a carrier material in a non-covalent binding in which the carrier material can be present either as a solid phase e.g. in the form of tubes, microtitre plates or polystyrene beads or in the form of particles e.g. latex particles. The fusion protein consists of an immunologically active substance which is necessary for the method and a binding peptide which mediates binding to the carrier material. All immunologically active substances which can be produced by genetic engineering e.g. antibodies and their fragments as well as antigens, e.g. HIV or hepatitis antigens or haptens such as hormones, drugs etc. are suitable for the method according to the present invention. The same peptides as described previously are suitable as the binding peptide. A material which is suitable for use in an immunoassay as a solid phase or as a particle and which can interact intermolecularly with the binding peptide i9 used as the carrier material . Carrier materials whiah are suitable for this are known to the expert. The process for the production of the fusion protein i8 carried out as described above. ~he solid phase-bound or particle-bound receptors produced in this way can be used for immunoassays of the heterogeneous and homogeneous type in a well-known manner. The procedure for these immunoassays is known to the expert and does not need to be described here in detail. Basically in this process the sample solution is reacted with at least two receptors whereby in the case of the heterogeneous immunoassay one receptor is bound to a ~ V ~ a solid phase and the other is labelled and in the case of the homogeneous immunoassay one receptor is particle-bound and the other can bind to the substance to be detected and to the particle-bound receptor. Complexes form between the receptors and the substance to be detected which result in a change in signal which, in the case of the heterogeneous immunoassay, is due to the label which can be an enzyme, or a fluorescent, chemiluminescent or radioactive substance and, in the case of the homogeneous immunoassay, is due to agglutination.

The invention is elucidated by the following figures and examples:

Fig. 1 shows the construction of the ~-glucosidase-Arg6 expression vector;
Fig. 2a shows the plasmid map of pKK177-3/GLUCPI_ARG6;
Fig. 2b shows the nucleotide sequence of the plasmid pKK177-3/GLUCPI ARG6;
Fig. 3 shows a diagram of a maltose biocatalyser with complete substrate conversion; substrate buffer:
10 mM KPP, 1 mM EDTA, 6 mM maltose, pH 7.0;
Fig. ~ shows a diagram of a maltose blocatalyser with substrate conversion limited by substrate saturation substrate buffer: 10 mM KPP, 1 mM EDTA, 0.15 M
maltose, pH 7.0;
Fig. 5 shows the renaturation kinetics of ~-glucosidase-Arg6 denatured on a carrier Denaturation buffer: 10 mM KPP, 1 mM EDTA, 2 mM
DTE, 6 M urea, pH 6.8 7 ~ ~ 3 Renaturation buffer: 10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 6.8 .

E x a m p l e s Standard methods wer~ used to manipulate DNA such as those described by Maniatis et al., (1982) in Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724. The molecular biological reaqents which were used were employed according to the manufacturers' instructions.

Materials:
Restriction endonucleases and T4-DNA ligase were from Boehringer Mannheim GmbH; yeast extract, Bacto Trypton and Casamino acids were from Difco.

~acterial strains:
The E. coli strain HB101 (Maniatis et al., 1982, Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or GM~8 (Yanisch-Perron et al., 1985, Gene 33, 103-119) for the preparation of unmethylated DNA, were used as recipient strains for the plasmid amplification. The E. coli strain RM82, a methionine revertant of ED8654, (Murray et al., 1977, Mol. Gen. Genet. 150, 53-61), which contains (i) the R-plasmid pREM6677 (pePA119, DSM 3691P) which co~es for the lacIq repressor and for trimethoprime resistance and (ii) the ~-glucosidase PI-Arg6 expression plasmid pKK177-3/GLUC ARG6 (ampicillin resistance), was used for the expression of the ~-glucosidase-PI-Arg6 fusion protein. This strain is denoted RM82 Iq/pKK177-3/GLUCPI_ARG6 in the following.

~ 3 E x a m p l e Construction of the ~-glucosidase-Arg6 expression plasmid pKK177-3/GLUCPI-ARG6 The structural gene of the ~-glucosidase PI from baker's yeast was extended at the 3' end by a DNA fragment which codes for the amino acid sequence GlyArgArgArgArgArgArg.
In this way an ~-glucosidase PI fusion protein forms which contains 6 additional argini~e residues (polycationic ankering sequence) and a glycine as a "spacer" at the C-terminus.

For this purpose the ca. 4.7 kbp long plasmid pKK177-3/GLUCPI (production and description cf: EP-A O 300 425, Kopetzki, Schumacher, Buckel, 1989, Mol. Gen. Genet.
21~, 149-155) was partially digested with the restriction endonuclease EcoRI and completely digested with BclI. The synthetic DNA fragment 5'-AATTATGACGATATCC-3' 3'-TACTGCTATAGGCTAG-5' M~tThrIleSer....

was ligated lnto the isolated ca. 4.7 kbp long BclI/EcoRI vector fragment (construction: pKX177-3/GLUCPI SD) which destroys the EcoRI and BclI
-restriction cleavage site. The ca. 300 bp long EcoRI
fragment was isolated from the plasmid YRp/GLUCPI (DSM
4173P~ (production and description cf: EP-A O 323 838;
Kopetzki, Buckel, Schumacher, 1989, Yeast 5, 11-24).
This was re-cleaved with HinfI and the ca. 90 bp long EcoRI/HinfI fragment was isolated.

~ O ~ !~7 2 ~ ~

Afterwards the ca. 90 bp long EcoRI/HinfI fragment as well as the synthetic DNA fragment IleTyrLeuValLysGlyArgArgArgArgArgArgEndEnd 5'-AATCTACCTGGTCAAAGGGCGCCGACGTCGCCGGCGTTAATA
3'-GATGGACCAGTTTCCCGCGGCTGCAGCGGCCGCAATTATTCGA-5' ______ _____ HinfI HindIII

were inserted via a three-fold ligation into the ca. 2.85 kbp long EcoRI/HindIII-pKK177-3 vector fragment (DSM 3062) (construction: pKK177-3!EH).

The ca. 130 bp long EcoRI/HindIII fragment was isolated from the plasmid pKK177-3/EH and ligated into the ca. 4.65 kbp long EcoRI/HindIII-pKK177-3/GLUCPI-SD
vector fragment (construction: pKK177-3/GLUCPI ARG6).
The correct construction of the a-glucosidase-Arg6 gene was confirmed by restriction analysis and DNA
sequencing. The construction oP the ~-glucosidase-Arg6 expression Vector is shown diagrammatically in Figure 1.
In order to express the ~-glucosisase-Arg6 fusion protein, the plasmid pKKl77-3/GLUCPI ARG6 (Figure 2a and 2b, SEQ ID No:1) Was transformed into the E. coli strain RM82 Iq (ED Iq, DSM 2102).

7 ~

E x a m p 1 e 2 Expression of the ~-qlucosidase-Arq6 fusion protein in E. coli The E. coli strain RM82 which has the ~-glucosidase-PI-Arg6 expression plasmid and a R plasmid which codes for the lacIq repressor as well as for trimethoprime resistance was used for the expression of the ~-glucosidase-PI-Arg6 gene. The e~pression of the ~-glucosidase is under the control of the tac-hybrid promoter.

Culture conditions 500 ml modified M9 minimal medium (6 g Na2HPO4/1, 3 g KH2P04/1, 0.5 g NaCl/l, 1 g NH4Cl/1, 2.5 mg thiamine/l, 5 g Casamino acids/l, 20 ml glycerol/l ~87 %), 0.25 g MgSO4 7 H2O/1, 50 mg ampicillin/l, 10 mg trimethoprime/l) was inoculated with 1 % of an overnight culture of the E. coli strain RM82-Iq/pKK177-3/GLUCPI ARG6 (which was also in the above-mentioned medium). The cultures were incubated at 30C while shaking constantly. The growth was monitored by measurement of the optic~l density at 550 nm (OD550nm).

Induction of the ~-glucosidase After a cell density of OD550nm 0.7 - 0.9 was reached the formation of ~-glucosidase was induced by the addition of lactose (final concentration 0.5 %). The cells were harvested by centrifugation after ca. 16 hours.

~ s 2 ~3~

The increase in the ~-glucosidase content in the cells was monitored with the aid of 1 ml culture samples which were taken at different times after the induction and analysed.

Determination of the ~-qlucosidase activitv The cell pellet from 1 ml E. coli culture was resuspended in O.5 ml 10 mM phosphate buffer, 1 mM EDTA, pH 7.0, the cells were lysed by m,eans of ultra sound, the cell debris was separated by cell centrifugation and the supernatant was processed further as the cell lysate.

50 ~l of the supernatant (dilute if necessary~ was mixed with 3 ml potassium phosphate buffer (lOOmM, pH 6.8) which contained the ~-glucosidase substrate p-nitrophenyl-~-D-glucopyranoside (p-NPG; 2 mM). The ~-glucosidase liberates p-nitrophenol by enzymatic cleavage. This was determined by the increase of absorbance at ~05 nm.

E x a m p 1 ~ 3 P~oductio~ of th~ b~oç~talyser Cell lYsis and isolation of the crude extract The cell pellet from 500 ml E. coli culture was resuspended in 30 - 50 ml potassium phosphate buffer (KPP), l mM EDTA, pH 7.0, mechanically lysed by means of a French press (2 passages at 14000 psi; 1 psi= 0.068 atm) and subsequently the cell debris fragments were ' 2 3 ~

separated by centrifugation (10 min, 7000 rpm). The cell lysate obtained in this way was used directly for the production of the biocatalyser.

carrier materlals The cation-exchanger Fractogel~ EMD S03 -650 ("tentacle-type gel") was used whose exchanger groups are freely mobile in space on flexible polymer chains. By this means a good interaction of the c~lumn material with the proteins to be bound is ensured.

The cation-exchanger Fractogel~ EMD S03 -650 was eguilibrated with 10 mM potassium phosphate buffer, 1 mM
EDTA, pH 7Ø As an alternative heparin-Sepharose~ CL-6B
was used as the gel matrix. The binding capacity for the ~-glucosidase-Arg6 fusion protein was determined as 3000 U/ml for both gel materials.

Determination of the extent of loading In order to immobilize the ~-glucosidase-Arg6 fusion protein on Fractogel~ EMD S03 -650 or Heparin-Sepharose~
CL-6B, the crude extract (a-glucosidase activity ca.
200 U/ml) was applied to the column at a pumping rate of ca. 10 column volumes/hour. Afterwards the column was washed with 10 mM potassium buffer, 1 mM EDTA, pH 7.0 and the loading of the column material with ~-glucosidase-Arg6 fusion protein was determined. For this the total activity of ~-glucosidase which was not bound to the column was measured in the column eluate (eluent and washing buffer) and subtracted from the amount of enzyme applied to the column. The binding of the ~7~3~

~-glucosidase-Arg6 fusion protein to Fractogel~ SO3 -650 or heparin-Sepharose~ CL-6B was 95-98 %.

E x a m p l e 4 Conversion of substrate In order to test the function of the ~-glucosidase catalyser, the hydrolytic properties of the enyzme were determined with respect to the ~-~lucosidase substrates maltose and p-NPG. The amount of glucose released from maltose, or the nitrophenol released from p-NPG, per unit time by the a-glucosidase catalyser is a measure for the catalyser performance. The release of p-nitrophenol from p-NPG was determined photometrically in the column eluate (analogous to the ~-glucosidase determination). The hydrolysis of maltose was monitored in the column eluate via the release of glucose.

Glucose determination Test principle:

hexokinase D-glucose ~ ~TP -~ > glucose-6-P + ADP

glucose-6-phosphate dehydrogenase glucose-6-P + NADP+ --------------> gluconate-6-P
+ NADPH+H+

6 2 3 ~

Test procedure:

625 ~1 triethanolamine buffer (0.3 M TRA, 4 mM MgC12, pH 7.5) + 50 ~1 ATP/NADP solution (150 mM ATP, 12 mM NADP in the above-mentioned TRA buffer) + 50 ~1 sample containing glucose Read the absorbance at 366 nm (A1) + 10 ~1 enzyme mixture (glucose-6-phosphate dehydrogenase 1 mg/ml and hexokinase 1 mg/ml; each in 3.2 M ammonium sulphate solution).
After the reaction has reached completion A2 is read at 366 nm.
The glucose content of the sample is calculated from the difference A2 ~ A1.

Maltose hydrolysis by means of non-covalently immobilized a-alucosidase Maltose bioreactor with çomplete substrate conversion 2500 U ~-glucosidase-Arg6 was immobilized on a heparin-Sepharose~ CL-6B column (ca. 3 ml column volume) according to the procedure described in Example 3. The substrate buffer contained 6 mM maltose; the pumping rate was 0.5 ml/min.
In continuous operation, the hydrolysis of maltose was observed over a time perîod of 33 days (Figure 3).
Result: The reactor showed 100 ~ substrate conversion during the entire period of observation.

~ o a~ r~ 2 -~ ~

Maltose bioreactor with limited substrate conversion 180 U ~-glucosidase-Arg6 was immobilized on a Fractogel~
EMD S03 -650 column as described in Example 3. In order to compare the substrate conversion and the binding stability at different temperatures, a reactor was operated at room temperature (RT) and a further reactor, which was loaded identically, was operated at 30C.

The substrate buffer contained 0.15 M maltose; the pumping rate was 5 column volumes/hour. The maltose conversion of the bioreactor was observed over 30 days.
During this time period a decrease from initially 50 %
to 38 % (30C) or from 36 % to 28 % (RT) was observed (Figure 4).

E x a m p l e 5 Renaturation of denatured ~-glucosidase-Arq6 fusion prote~ on the carrier Purified a~glucosidase-Arg6 fusion protein (ca. 80 U/mg) was denatured in 10 mM potassium phosphate buffer, 1 mM
EDTA, 6 M urea, 2 mM DTE pH 7.0 and applied to a "tentacle-type gel" column equilibrated with denaturing buffer. After application of the sample, the column was rinsed with renaturing buffer (lO mM potassium phosphate (KPP), 1 mM EDTA, 2 mM 1,4-dithioerythritol (DTE), pl~ 7.0) (ca. 10 column volumes). The bound and renatured ~-glucosidase-Arg6 fusion protein was eluted with 10 mM
potassium phosphate buffer, 1 mM EDTA, 1 M NaCl, pH 7Ø
In the eluate obtained with 1 M NaCl, a specific ~-glucosidase activity of ca. 20 U/mg could be detected.

~ii 6123 ~ x a m p l e 6 De- and renaturation of native ~-alucosidase-Ara6 bound to the carrier The carrier ma~erial Fractogel~ EMD SO3--650 was loaded in a batch procedure with purified ~-glucosidase-Arg6 fusion protein (ca. 800 U), filled in a column, rinsed for one hour with denaturing buffer (10 mM KPP, 1 mM
EDTA, 2 mM DTE, 6 M urea, pH 6.8) and subsequently washed with substrate buffer (10 mM KPP, 1 mM EDTA, 0.15 M maltose, pH 6.8).

The renaturation of the ~-glucosidase can be monitored in the column eluate by the increase in the glucose released from maltose (Figure 5).

E x a m p 1 e 7 Immunoloqical determination of anti ~IV antibodies usina an antiqçn bound to a solid ph~se HIV ~nti~~n The HIV polypeptide HIV2(envgp32)-~IVl(poly32-envgp41-gagpl7-p24-15-poly(Arg-Lys) is a "multifunctional" HIV
fu.sion polypeptide produced by means of recombinant DNA
technology. It consists of protein partial regions of the gag, pol and env region of HIVl and of the env region of the HIV2 retrovirus. In addition the HIV
polypeptide has at its C-terminal a polycationic anchor sequence consisting of 13 positively charged amino acids (arginine and lysine).

~A7~3~

The protein sequence of the HIV polypeptide is shown in SEQ ID NO:2.

Expression of the HIV fusion protein in E. coli The E. coli strain RM82+, DSM 5446 (a lactose revertant of RM82) which contains the HIV fusion protein expression plasmid pKK233-2/MYYL_gp32_polp32_gp41_p24-polyArgLys (ampicillin resistance) and the dnaY-lacq plasmid pUBS500 was used for the expression of the HIV
fusion protein.

The construction of the HIV expression plasmid is described in the German Patent Application P 40 02 636.1 dated 30.01.1990.
pUBS500 (EP-A 0 373 365) is a pACYC derivative (Chang and Cohen, J. Bacteriol. 134 (1978) 1141-1156) which in addition to the kanamycin resistance gene has a lacIq repressor gene (Carlos, Nature 274 (1978) 762-765) and a gene for the t-RNA arginine (anticodons: AAG, AGG) which is rare in E. coli.

The recombinant E. coli cells were cultured at 30C in DYT medium (16 g bactotryptone, 10 g yeast extract, 5 g NaCl per liter) supplemented with 50 mg/l ampicillin and 50 mg/l kanamycin. After reaching an optical density of 0.6 - 0.8 at 550 nm the cells were induced with IPTG
(isopropyl-~-D-thiogalactopyranoside, final concentration 1 mM). After an induction time of 4 - 10 hours the cells were centrifuged, washed with 10 mM
Tris-HCl buffer, pH 7.0 and stored at -20C until further processing.

~B~72~3 Cell lysis, solubilization of the HIV fusion proteln 20 g (wet weight) E. coli cells were resuspended in 400 ml 100 mM Tris-HCl buffer, pH 6.5 - 7.5, 0.25 mg/ml lysozyme was added and incubated for 0.5 - 1 hour at room temperature. Afterwards the suspension was cooled to 0 - 4C and the cells contained therein were lysed by ultrasonication (French press). The cell debris and the insoluble aggregated HIV fusion protein ("inclusion bodies") were separated by centrifugation and the pellet was washed 1 - 2 times with 200 - 400 ml 0.5 M NaCl or KCl and 1 ~ (v/v) Triton-X-100. Subsequently the pellet was resuspended in 20 ml 50 mM Tris-HCl pH 8.0 containing 8 M urea and 5 mM ~-mercaptoethanol at room temperature for 1 hour while stirring (magnetic stirrer) and the insoluble cell components were removed by centrifugation. The protein concentration of the solubilized proteins was determined according to Bradford (Anal. Biochem. 72 (1976) 248-254) modified according to Gotham et al. (Anal. Biochem. 173 (1988) 353-358).

~IIVl (polp32-~nvqp41-q~qP17-p24-15-poly¢~q-L~s) protei~
on a ~el mat~l~

1 ml cation exchanger Fractogel~ EMD-S~3-650(M) equilibrated in 50 mM Tris-}lCl, pH 8.0 and 8 M urea was incubated with 100 - 200 mg solubilized HIV fusion protein at room temperature for 2 - 6 hours while shaking. The proteins which were not bound to the gel were removed by washing with equilibration buffer.
Afterwards the gel was re-incubated with 10 mg/ml bovine serum albumin in phosphate-buffered saline (PBS, 0.15 mM

~723-~

sodium phosphate; 0.9 % NaCl; pH 7.2) and washed with demineralized water containing 0.5 % Tween 20.

Determination of anti HI~ antibodies The sample (10 - 100 ~l human serum) was diluted one hundred-fold in PBS containing 10 % calf serum and incubated for 4 - 12 hours at 37C with 10 ~l gel which was coated with HIV fusion protein. It was subsequently washed three times with 1 - 1.5 ml washing solution (0.5 % Tween 20 in demineralized water).

In the second step it was incubated with a conjugate of peroxidase and polyclonal antibody (POD conjugate, ca. 30 mU peroxidase/ml in PBS) which was directed against the Fc ~part of human IgG and washed three times with washing solution.

Afterwards 1.5 ml substrate solution (1.6 mM 2,2'azino-di-[3-ethylbenzthiazoline sulfonic acid(6)]diammonium salt (~BT ~ ; 95 mM phosphate-citrate buffer, pH 4.4;
3.1 mM sodium perborate) was added, incubated for 15 -~0 min at room temperature and the absorbance in the centrifuged sample was determined at 492 nm as a measure for the amount of specific antibody present in the sample.

Normal sera showed a relative signal strength in relation to anti-HIV pool sera (oD492 anti-HIV pool serum/OD49Z normal serum) of 7 - 50 (oD492: absorbance at 492 nm).

Details of the deposition of the aforementioned cell lines are set forth below.

-- 2 5 -- i~ 7 2 3 ~
DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH

DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel.:t0551) 393822 _ Boehringer Mannheim GmbH Date Biochemica Werk Tutzing 7.5.1g86 Postfach 1263/64 8132 Tutzing ACKNOWLEDGMENT OF RECEIPT

We acknowledge that the plasmid with the identification reference pePa 119 given by the depositor was deposited on the 9.4.1986 in the German Collection of Microorganisms under the receipt number DSM 3691P.

GERMAN COLLECTION
OF MICROORGANISMS

- 26 ~ 2 ~ ~3 DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH

DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel.:(0551) 393827 Boehringer Mannheim GmbH
Biochemica Werk Tutzing Abt. E - B 1 Postfach 1263/64 8132 Tutæing Your ref. Our ref. Date 38687 14.07.1987 ACKNOWLEDGMENT OF RECEIPT

We acknowledge that the plasmid with the following reference number YRp-Gluc.pI

given by the depositor was deposited on the 29.06.1987 in the German Collection of Microorganisms under the receipt number DSM 4173 P.

GE~MAN CO~LECTION
OF MICROORGANISMS

- 27 - ~li 72 ja DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH
DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel (0551)393827 Boehringer Mannheim GmbH
Biochemica Werk Tutzing Abt. E - B 1 Postfach 1263t64 8132 Tutzing Your ref. Our ref. Date Bk-Bl 39087 14.7.1987 Use of a DSM strain in a patent application Confirmation We hereby confirm that the microorganism mentioned below is present in the general collection of the DSM. The strain will be stored for at least a further 30 years from the date of this confirmation. The term is calculated from the date of publication if the strain was referred to when the patent was applied for and the DSM was informed of this.

In addition the culture will be stored for a term of 5 years after the last application for delivery of a sample has been received.

Samples of this culture which are capable of reproduction were and will be issued to anyone during the entire period according to the national regulations for trading with microorganism~.

An examination of the viability was carried out on the date mentioned below. The micxoorganism was viable at this time.

Taxonomic designation DSM Date of Date of Date of patent for the microorganism number accession viability publication in the DSM test according to the applicant Escherichia coli HB101 1607 16.7.1979 9.3.1987 Micromonospora 43141 15.4.1975 10.7.1987 echinospora German Collection of Microorganisms BUDAPEST TREATY OU THE INTERNATIONAL
~ 2 8 - R~COGNITIOH OF THE DEPOSIT OF MICROORGANISMS
COR THE PURPOSES OF PATENT PQOCEDURE ~ f~

INTERNAT~ONAL FORM

Boehringer Mannheim GmbH
Biochemica W~rk Tutzing Abt. E--Bl RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Postfach 1263/64 issued pursuant to Rule 7 1 by the INTERNATIONAL DEPOSITARY AUTHORITY
D--8132 Tutzing identified at the bottom of this poge ~AME AND ADDRESS
OF DEPOSITOR
L J

~.......... .
Idont1fleation reforonce ~iven by the Accession number given by the DEPSIToR BNTU INTERNATIONAL DEPOSITARY AUTHORlTr 54-2/pKK 177-3 DSM 3062 .
Il SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOHIC DESIGNATION
. __ The microor9ani8m ldontified under I abovo ~as accompanied by ) a sciontiflc dascrlption tXl a propos d taxonomic do4i~natir~n tH~rk ~ith a cro8s uare npp~icabla) _ ~
Ill RECEIPT AND ACCEPTANCE
Thl~ Intornational D~poaitary Author'ty occ~pta tho microor~ni8m idQntitiQd under I obovo uhlch ~a8 rocoivad by it on 2 6 - O 9 - 1 9 8 4 ~dat~ o~ th- or~g~nn p ._ _ ........ _ _ .. _ .
IV INTERNATIONAL OEPOSITARY AUT~ORITY
. .. . __ __ ~
NaT~ GERHAN COLLECTION Signatura~) ot p r~on~) hnving tha po~or OF MICROORG~NISHS to roproaant tho Intarnatlonal Depository Addrass Grl~abachntr~n- G Authorlty or ot authorl~ed o~tlclol~) D_3400 Go~tlngan . 3-10-1984 _ .
1 ~hero Rulo 6 4~d) opp~ies such dote is tho date on ~hich the status of international depositary authority ~as acquired ~here a doposit moda outsid- tho 8udapest Trenty ofter the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty such date is the date on ~hich the microorganism ~as received by the intern~tiona~ depositary outhority Form BP/4 ~sole page) 1081 BUDAPEST TREATr ON TNE INTERNATIONAL
- 2 9 - RECOGNITION OF THE DFPOSIT OF MlCROORGANlSMs ~ O ~ ~ ~ 3 FOR THE PURPOSES OF PATENT PRocEDuRE

INTERHATIONAL FORM

Boehringer Mannheim GmbH
Postfach 120 RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7 1 by the 8132Tutzing INTERNATIONAL DEPOSJTARY AuTHoRlTr identified st the bottom of this page NAME AUD ADDRESS
OF DEPOSITOR
L J
. . ~
I IDENTIFICATION OF THE MICROORGA~ISH
_ ,....... _ .
Identificstion reforoneo given by the ~ccossion numbor given oy tho DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY

. DSM 2102 - _ Il SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOH C DESIGNATION
._ The microorgonism 1dent1fled undor I abovo ~D8 accomponied oy ) o se10ntifie doserlptlon X) e proposed toxonomie designotion ~Hork ~1th o eros~ ~ore npplienblo) ~-- - ...
Ill RECEIPT AND ACCEPTANCE
_ _ ,_ __ _ . .
This Intornat10nnl Dopositnry Author1ty oecopt8 tho microor90nl8m Idantlt1Qd unchr I ooovo ;
uh~ch ~8 roco~vod by ~t on 1 - 1 0 - 1 9 8 1 ~cbt~ ot tho or~9~n~ d p ~ ) ,_ . . ,_ . . .__ _ - _ _ _ _ IV INTERNATIONAL DEPOSITARY AUTNORITY
. _ .......... ,= ,,_ . _ . _ _ __ NQme~ GERHUN COLLECTION Si~noturo~) ot p rnon~) h~vlng tho po~-r OF MICROORGANI5HS to reprosQnt tho Intornat10nol Dopo~ltnry Addrosr Grlgooqeh~tro~no ~ Authority or of cuthor1~eci offic1al~s) D_3400 GoLt1ngon ~ .C~t ~ Dnto 2-06-1982 . _ I ~horo Rulo 6 4(d) applies such date is the date on ~hich the stotus of international depositnry authority ~a~ acr1uireci;
~horo a deposit mado outsido the SudDpest Tre~ty ofter the acquisition of the status of internntional depositary authority is convorteci into a deposit urder the Budepest Treaty such date is the date on which ~he microorganism ~as received by the internotional depositary authority Form BP/4 ~sole page) 1081 . . _ _ _ .

~ ~ l 7 2 ~,3 SEQ ID NO:1 TYPE OF SEQUENCE: Nucleotide sequence LENGTH OF SEQUENCE: 4648 FORM OF STRAND: single strand Plasmid Pkkl77-3/GLUCPI_ARG6 181 ~1~1~ G~a~ CA~ICG ~1~ (~1~ GCGG~CA

241 ~a ~a;c~ ~CGAT A~G~ (~GMACAG AZ~CC~

421 ~ ~1~1~ CGrm;~ Cl~l~CA~ CA.:Ga~ ~T

541 l~ Cl~ Gl~ CAI~ ~1~ ACCN~

661 Gll~l~l~ A ~ CC~ ~aG6~ A C ~ A ~ l~ C ~ AC~

721 Ir~r~LXTlr~F~XT~ GTn~qIG G~rcT~T GA~K~YPA ~DG~TTA

781 CCTCCG~ T~CG~GTC GDCh~T~A CTnUY~T~G GAGuY~aG A~D3~PG

841 G3C~5TT GAY~Y3G ID~G~IG GC~3~XP~ G3~YaIG GTTT~A~

901 CGr~r ~ CG~ q~ G~ ~aCAA

i 7 2 .....3 961 AACCTCG~AA ~CAP~a~ CAAA~I~ Gl~l~C:~T G(~CCI~GA ~A

1021 ICP~AA C~C~aG~T TI~A~AA CA~l~AA G~ AAP~A~

1081 AGICGGl~A Gl~ G~aG~AA ~C Al~aGlOE~ ~ACG~

1141 AGI~;CG~A GITll~CCI~ I~CGCP~QI~ 1~1~ ACCICGCC~ TlTICCGl~A

1201 ~A~ C~ ~A~ G~AA~ A~OEA~G~ ACrITl~T

1261 ~1~(3Gl~ ACl~l~ (~ CI~G:G APIOEG~C A~;CCCG~C

1321 A~CGA~ m~;;CG Al~CGCCA~ e~Aa A~A A~I~AC

1381 A~Ga~ ~1~ ~CGl'r ~I~T C~I~G A(i~CA

1441 G~l~aA~ A~A~ C~AAA Gl~ Grl~A AaAACA~A

1501 CG~ A~AAMI~l~ Tl~ Cl~;A A~IT TrlTl~A~

1561 Ah~A Crl~Gl:G Al~l~G~ AU~I~al~ CC~ACGA A~G~C

1621 C~l~A IT:I~CC CAG~I~A A(rl~lTr Iq~l~ AA~TI~A

1681 ~A~ A~ 1~3;~C AOE~G AS ~ IW~I~ AITm~;AI~

1741 AA~Clq~; C~ACAA AGAI~A ~ Al~l~ ACGAll~CA

1801 A~ TI~ C~I~ At~ Tll~l~Cr AMG;a~:; G~AAG~

1861 ~mI~ ~1~;~ 1~GI~3GCGA AGA~ I~AI~C CA~AGa~G

1921 ~mA ~11~ I~A ~s~ G~CGIITCCT CCAGA6rm 1981 G~ACCl~IX3G G~AA ~9a~I~ CA~GCGC CG~C~CGCC GGCGl~A

2041 AGC~ll~IGlT TmGCGGAl'G AG~G~AG~ Tl~l&A ~Ca~TTAA AI~CGC

-~ 3 2101 ~LaPGCYG~ qGa~A~Z~ GaAITrG~CT GECGaC~GI~ GCGCGETa~T CC ~ C

2161 CCCATGCCGA ALICAEPAEr GAAACGCCGT AGCGCCGAIG GIPGrGnaGG GTCTCCCCAT

2221 GCGAEAGIAG GGAALTGCCA GGCA~CAAAr AAAP~GAAAG GC~X~GTCGA AAG~LqGEGC

2281 cmcGTTTT P~CqGTnarT TGTCGG~G~A CGCTCTCCTG AEn~GGaCAA ATCCGCCGGG

2341 AGCGG~ITTG A~CGTTGCGA ~ CGGCC CGGAGGaTGa CGGGCALGaC GCCCGCCAIA

2401 AaCTGCCAGG CA~CAAArT~ AGCAAaYGC CArGCIGaDG Ga~rGOCm TIscGrTTcT

2461 A~aaAclcsr TTGTTT~m TTCqAAA~AC ATlCaAArEr GTa3CCGrTC A~GaGa~AAr 2521 AaCCCTGaT~ AA5GCTICAA TPP~ATlraA AAAGGaAaaa TATGaGTa~T CPALAT51CC

2581 GTGrCGCCCT TAlTcccm ~ LGGCAr mGccTlcc IGTTTTTGCT CACCCAGAAA

2641 CGCTGGT~aa PGTPAAAGAT GCTGh GA5C h~ L ACG~GTGGGT TPL~IOEaAC

2701 TG34ICTCAA CAGCGG~AG ATCCrr~AEA Gl m CGCCC CGAAGAACGr m CCAE~A

2761 TGAGCAC m TAAE3TIe5G CrATGTGGCG CGGrATTAlC CCGTGil~aC GCCGG~CA~G

2821 AGCAACICGG TCGCCGCArA CACraq~lCrC AG~ADGACrT GGTIGAGTAC TCACCAGTC~

2881 C~GAAAAGCA TcTTAcGGAT GGCA~GACaG TAhGhGAATT ATGCAGTGCT GCC~IAALCA

2941 TGKGIGATAA CACra~GGCC AE~rrYCT~C 'r~AAOGAT CGG~GGACCG AE GaGCTAa 3001 CCLrTI~ m G~ACAACAIG C~ 5~P~G TAE~n3GCCT TGEn~GrIGG GAACCGGAGC

3061 rrGAA3~aAGC CATACCaaAC GACG~GCGTG AC~CCALCaT GCCrx AGcA ATGG AACAA

3121 CG`TTGCGCAA ACTATTAACT GGCGAACI~C TTACrCTaGC TrCCCGGCAA CAErrAAr~G

3181 ACra~AIGGA GGCGGArAAA GTTGCAGG~C CACrr-TGCG CTCGGCCCrr CCGGCTGGCT

s~ ~

3301 ~ ~ I~CCG~CG ~1~ CP~GGGG A~

3361 C~P~ ACG~A~ ~G

3421 M~l~l~a CCaal~ll~ ~T~C qT~G~G~ TI~AaAC

3481 ~AA~ C~I~AG Anrl~ P~a~ GPCC~ CCTI~ACGI~

3541 A~lll~CGl~ CC;Q~;;~G ~G;CCCCG q~G~a~ C~A;i~ ~1~

3601 cmTm~ Gt~GC~IC ~1~1~: AA~A~ A~CGCI~ CCAGCGGI~;

3661 I~lTL~ ~:5~aG C~X:A~

3721 C~CC ~aP3~ Cl~l~ IY~ ~cl~ac ~

3841 ~a~ Al~;CGC ~C

3901 GGDCGGGC5G AACGGGGGGT TCGnGCALaC PGlrCaGCTT GG~GOGaACG ~ CCG

3961 AaL5GAGæ~ CCTPCAGCGT GAGCArTGAG AAAGCGCCAC GC~IOOOGAA GGGAGAApGG

4021 CGGACAG31A TCCGGTAA~C GacAGGGTcG GAACaGC~G~ GCGCALGAGG GAGCTTCCAG

4081 GGGEAAACGC CTGGr~5CTT TP~AGTCCTG ICGGGnT~CG CCACrICTG~ CTTGAGCGTC

4141 GATTTTT35G PIGCTCGTCA GGGGGGCGGA GCCIA5GG~A AAACGCCA~C AACGCGGCCT

4201 TTTIACGGIT CCTGGCCTTT IGCTGGCCTT TT~ITCACAr GTTCTTTCCT GCGTT~ICCC

4261 CTGaTlCTGr GGAI~ACCGT ATTACCGCCT TTGAGnGA~C TGAIACCGCT CGCCGCAGCC

4321 GAACGACCGA GCGCAGCGAG TCAGnGAGCG AGGAAGCGGA AGAGCGCCTG AIGCGGqA~T

~381 II~CCI'r~C ~:A~l~IOE GGI~P~lTI~C ACCOE~I~ GI~CI~IC P~aA~

4441 ~I~1~5~ C(~A~ P~ C~CICCGCI~ TCGCI~CGIG 1~1~

~1501 GGC~CGCCC CGPL~GC CA~CGC l'OEGCGCCC IGI~ Gl~l~CCC

4561 GGCP~CCGCI ~C1~P~ ~l~P~ CICCGGG~C TGC~IGI~C AQ~ll 4621 ACCGI~I~ CCG~CG CGZ~;GC~

SEQ ID NO: 2 TYPE OF SEQUENCE: Amino acid sequence LENGTH OF SEQUENCE: 770 amino acids FORM OF STRAND: single strand ~Set Tyr Tyr Leu Glu Phe Gln Gln Gln Gln Gln Leu Leu Asp Val Val 16 Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Lys Asn 32 Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala 48 Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr 64 Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn Met Thr 80 Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser 96 Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu 112 Leu Gln Lys Leu Asn Ser Trp Asp Asp Pro Leu Glu Ser Cys Asp Lys 128 Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro 144 Gly Ile Trp Gln Leu Asp Cys Thr HiS Leu Glu Gly Lys Ile Ile Leu 160 Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro 176 Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly 192 Arg Trp Pro Val Lys Val Ile HiS Thr Asp Asn Gly Ser Asn Phe Thr 208 Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu 224 Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met 240 Asn Lys Glu Leu Ly~ Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu 256 His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile HiS Asn Phe Lys 272 Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp 288 Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile 304 Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Leu 320 Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val 336 Ile Gln Asp Asn Ser Glu Ile Lys Val Val Pro Arg Arg Lys Ala Lys 352 Ile Ile Arg Asp Tyr Gly Lys Gln Ser Asp Arg Ser Ser Arg Val Gln 368 Thr Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu 384 Arq Ala Ile Glu Thr Gln Gln HiS Leu Leu Gln Leu Thr Val Trp Gly 400 Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln 416 Asp Gln Arg Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 432 Thr Thr Thr Val Pro Trp Asn Thr Ser Trp Ser Asn Lys Ser Leu Asp 448 Thr Ile Trp HiS Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp 464 Asn Tyr Thr Ser Ser Asp Lys Gly Asn Ser Ser Gln Val Se~r Gln Asn 480 Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val l~is Glll Ala Ile 496 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile G1U Glu Lys Ala 512 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 528 Thr Pro Gln A~p Leu Asn Thr Met Leu A~n Thr Val Gly G1Y E~is Gln 544 Ala Ala Met Gln Met Leu Lys Glu Thr Ile A~n Glu Glu Ala Ala Glu 560 Trp A~p Arg Val His Pro Val ~-~ic Ala Gly Pro I1Q Ala Pro Gly Gln 576 Met Arg G1U Pro Arg G1Y Ser Asp lle Ala Gly Thr Thr Ser Thr Leu 592 Gln Glu Gln Ile G1Y Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly 608 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 624 Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 640 Yro Phe Arg Asp Tyr Val Asp Arq Phe Tyr Lys Thr Leu Arg Ala G1U 656 (;ln Ala Ser Gln G1U Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 672 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 688 Ala Ala Thr Leu Glu G1U Met Met Thr Ala Cys Gln G1Y Val Gly Gly 704 Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr 720 Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Lys 736 Lys Thr Val Lys Cys Phe Asn Cys Gly Lys G1U G1Y His I le Ala Lys 752 Asn Cys Arg Ala Ser Arg Lys Lys Arg Arg Arg Lys Lys Arg Arg Lys 768 Lys Lys 770

Claims (18)

C l a i m s

1. Process for the enzymatic treatment of substrates, w h e r e i n the substrate to be treated is brought into contact with a biocatalyser which was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA
fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and the enzymatically treated substrate is subsequently isolated.

2. Process as claimed in claim 1, w h e r e i n a peptide which is mainly composed of positively charged amino acids is used as the binding peptide.

3. Process as claimed in claim 2, w h e r e i n a peptide which is mainly composed of the amino acids lysine and/or arginine is used as the binding peptide.

4. Process as claimed in claim 1, w h e r e i n a peptide which is mainly composed of negatively charged amino acids is used as the binding peptide.

5. Process as claimed in claim 4, w h e r e i n a peptide which is mainly composed of the amino acids aspartic acid and/or glutamic acid is used as the binding peptide.

6. Process as claimed in claim 1, w h e r e i n a peptide which is mainly composed of the amino acid histidine is used as the binding peptide.

7. Process as claimed in claim 1, w h e r e i n a peptide which is mainly composed of the amino acids alanine, valine, leucine, isoleucine, phenylalanine, tryptophan and/or tyrosine is used as the binding peptide.

8. Process as claimed in one of the previous claims, w h e r e i n the binding peptide has in addition a spacer of 1 to 10 amino acid residues at the point of linkage to the biologically active substance.

9. Process as claimed in claim 8, w h e r e i n the spacer has 2 to 4 amino acid residues.

10. Process as claimed in claim 8 or 9, w h e r e i n the amino acids proline, glycine, alanine and/or serine are used for the spacer.

11. Process as claimed in one of the previous claims, w h e r e i n the end of the fusion protein which carries the binding peptide is protected by a non-charged amino acid (preferably proline) which is fused to it.

12. Process as claimed in claim 11, w h e r e i n proline is used as the protective amino acid.

13. Process as claimed in one of the previous claims, w h e r e i n a material which has negatively charged groups is used as the carrier material.

14. Process as claimed in one of the claims 1 to 12, w h e r e i n a material which has positively charged groups is used as the carrier material.

15. Process as claimed in one of the claims 1 to 12, w h e r e i n a material which has complexed metal ions is used as the carrier material.

16. Process as claimed in one of the claims 1 to 12, w h e r e i n a material which has hydrophobic groups is used as the carrier material.

17. Method for the detection of specifically bindable substances according to the heterogeneous immunoassay principle using a receptor bound to a solid phase, w h e r e i n the sample solution as well as at least one labelled receptor which is capable of binding to the substance to be detected are brought into contact with a solid phase-bound receptor which was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate which contains the fusion protein into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interactions and after incubation the solid phase is separated from the liquid phase and the label is determined in one of the two phases.

18. Process for the detection of specifically bindable substances according to the principle of the homogeneous immunoassay using a particle-bound receptor, w h e r e i n the sample is incubated with at least one particle-bound receptor which is capable of binding to the substance to be detected, which receptor was obtained by combining, in order to produce a fusion protein, a gene which codes for a biologically active substance and a DNA fragment which codes for a binding peptide which can interact with a carrier material then inserting the combined gene and DNA fragment into a suitable vector, transforming it in a suitable organism, culturing the organism, lysing the cells and bringing the lysate which contains the fusion protein into contact with a carrier material which is capable of binding the binding peptide whereby the fusion protein binds to the carrier material by intermolecular interaction and after incubation the solid phase is separated from the liquid phase and the agglutination is determined after incubation.