DE10352366A1 - Vector system that includes transcribable gene and pH-sensitive reporter gene, useful for identifying enzymes that catalyze pH-dependent reactions, also derived expression and substance libraries - Google Patents

Abstract

Vector system (VS) comprising at least one vector (V) that includes at least one target gene (TG), transcribable from a promoter, and at least one pH-sensitive reporter gene (RG), transcribable from the same, or some other, promoter, is new. Independent claims are also included for the following: (1) expression system (ES) comprising a host cell, or culture, transfected with VS; (2) substance library that contains VS; (3) expression library that contains ES; and (4) identifying target gene products that modulate pH.

Description

The The invention relates to a biotechnological method for finding as well as for the determination of the catalytic activity of enzymes and for this Needed procedure Vectors and libraries.

Of the Use of enzymes in catalytic synthesis processes gains in the chemical industry increasingly important. For this reason be great Made efforts to identify new enzymes or the activity known enzymes by artificial changes to vary.

Next the conventional one Methods for the untargeted mutagenesis of proteins are becoming increasingly common used evolutionary methods for targeted variation of proteins. It will be first Libraries are generated on modified biomolecules, which subsequently the activity be tested by their individual members. Such evolutionary methods can also for improving biocatalysts with regard to their catalytic Properties are used. Here is the individual test the individual generated library members, in particular at Libraries of biocatalysts, as well as the subsequent sorting out the most active members from the large number of library members very time consuming and expensive.

One simple assay for determining the catalytic activity of individual Enzymes is an agar plate assay. This is in the nutrient medium one for given the reaction detection specific indicator. Own one Clone the sought enzymatic activity, so it is at the appropriate Place the agarose plate induces an indicator reaction. Indeed this method is limited by the fact that only certain, with the breeding ground Compatible, indicator reaction are applicable, especially pH-dependent detection methods can only be used very conditionally. In addition, such is one Detection method not feasible in a liquid culture solution, since a targeted subsequent separation of colonies no longer possible is.

A size Number of enzymes catalyze reactions, directly or indirectly to a change of pH lead. In particular, enzymes of the E.C classes No. 1 (oxidoreductases), 2 (transferases), 3 (hydrolases) and 4 (lyases) belong to this group. For this There is a need for specific screening methods which a selection regarding the activity and selectivity enable these biocatalysts.

It Therefore, the task was to develop a method that the Locating biocatalysts and rapid screening of such Enzyme, allowing for their biocatalytic activity.

The Task is performed by vector systems, which are at least one, of one Promoter of transcribable target gene and at least one, of the same or another promoter of transcribable reporter gene, that for encodes a pH-sensitive reporter gene product.

The Target gene is characterized by the fact that it has a pH-influencing Gene product coded. Such target gene products are in particular oxidoreductases (E.C class 1), transferases (E.C class 2), hydrolases (E.C class 3) and lyases (E.C class 4).

Under The term hydrolases in particular includes all enzymes, the substrates catalytically split with consumption of water and / or release of water. It can Carbon-oxygen, carbon-nitrogen, carbon-carbon, Carbon-halogen, phosphorus-nitrogen, sulfur-oxygen or phosphor-oxygen-bonds linked or hydrolyzed. Preferred hydrolases are esterases, such as Lipases, phosphatases, phosphodiesterases, sulfatases, Nucleases, glycosidases, such as. B. α- or β-glucosidases or α- or β-galactosidases, peptidases, such as Serine, cysteine, aspartate or metal proteases, ether hydrolases, Amidases, amidinases or nitrilases.

Under the oxidoreductases are preferably dehydrogenases, the alcohols in aldehydes or ketones or vice versa aldehydes or ketones in alcohols and aldehydes in carboxylic acids or conversely, carboxylic acids convert into aldehydes. Furthermore, preferred oxidoreductases the monooxygenases, the alkenes in epoxides, ketones in esters, cyclic ones Ketones in lactones, thioethers in sulfoxides or alkenes in alcohols or dioxygenases that convert aromatics to their diols and peroxidases, the alkenes in epoxides, thioethers in sulfoxides or convert aromatics into their alcohols.

preferred Lyases include in particular the carbon-oxygen lyases, such as The dehydratases or the carbon-nitrogen-lyases, such as As the aspartases or carbon-halogen-lyases, such as. B. Dehalogegenases.

To the preferred transferases in the sense of this invention are in particular the aminotransferases and kinases.

Next the course occurring allelic forms of said enzyme genes are also their artificial mutant forms as target genes of particular interest. Among the Term target gene in the context of the present invention continue to fall Partial sequences of course occurring and artificial mutated enzyme genes. Preferred partial sequences are those which for the respective class of enzymes contain conserved nucleobase segments or such partial sequences encoding enzyme domains encoding an enzymatic activity have.

When Reporter gene is z. As the pHluorin, a pH-sensitive mutant form of the GFP (eg described in WO 98/36081 or in Miesenböck, G. et al., Nature 1998, 394, 192-195.) or a variant thereof. Under a pHluorin gene variant become nucleic acid fragments with a homology of more than 50%, in particular more than 70%, preferably more than 85%, based on the pH-sensitive light emitting pHluorin gene variants (WO 98/36081) understood.

One Advantage of the use of the pHluorine as a reporter lies in the nearly linear dependence the emission band ratio from pH in the range of pH 5 to pH 10. Due to this, the Influencing the pH by a target gene product, eg. B. the activity a hydrolase a specific substrate, to be evaluated very accurately.

Most preferably, a racemic mixture of the pH is used. The racemate shows two pH-dependent emission _peaks in spectrometric investigations, one at 395 nm and one at 475 nm. The ratio of the emission intensities R _475/395 changes reversibly depending on the pH. A great advantage of the racemic mixture of pHluorine is that not only the ratio of the emission peaks is a measure of the pH in the environment, but also that this ratio is independent of the pH of the fluorine itself. In determining the emission ratio, it is not absolutely necessary to measure the wavelength at the emission maximum; it may have a wavelength range from 360 nm to 440 nm, preferably between 380 nm and 410 nm, and from 450 nm to 500 nm, preferably between 460 nm and 490 nm , be used.

to Preparation of the vector systems according to the invention The target gene and the reporter gene can either be together in one Vector or in at least two equal or two different ones Vectors are cloned.

suitable Expression vectors are for. PGEM, pUC, pGEX, pTRX, pJOE or pQE for E. coli, but also expression vectors of other prokaryotic Protozoa can be used. As suitable expression vectors for yeasts have z. For example, the pREP vector, the pINT vector of the pTZα / β vector or the pTZsp vector proved. For the expression in insect cells are z. B. baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells are z. B. SV40 vectors, which are commonly available. Particularly preferred are expression vectors for unicellular pro- and eukaryotic Organisms, in particular from the group pGEX, pJOE, pREP and pINT.

The Vectors can in addition to the usual Markers, such as As the ampicillin resistance, other functional nucleotide sequences for regulation, in particular for repression or induction of expression of the target gene and / or the reporter gene. As promoters are preferably inducible promoters such. B. the rha promoter or the nmt1 promoter is used. Other proven promoters are z. B. the ADH-2 promoter for expression in yeasts (Russel et al., (1983) J. Biol. Chem. 2674), the baculovirus polyhedrin promoter for expression in insect cells (see eg EP-B1-0127839) or the SV40 early promoter or the LTR promoters z. From MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).

The expression vectors of the invention can additional functional sequence regions, such. B. an origin of replication, Operators, or contain termination signals.

An example of a vector system according to the invention is a mixture of the vectors pJOE 2792.1, containing as target gene the Pseudomonas fluorescens esterase (PFE) gene (Pelletier, I. and Altenbuchner, J. (1995); A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity. Microbiology 141, 459-68 ) and the vector pGEX_rat_pHluorin (Miesenbock, G., De Angelis, DA and Rothman, JE (1998), Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins, Nature 394, 192-195; see also 1 ). However, both expression plasmids can also be used to construct an expression plasmid which, in addition to the target gene, here the PFE gene, simultaneously contains the reporter gene, in the present case the pHluorin gene ( 2 ). The resulting expression plasmid is referred to as pGEX_pH_PFE.

With The vector systems described can be host cells with the conventional Methods, such. As the PEG / DMSO method or by electroporation, be transformed.

Thereby another object of the present invention is obtained these are expression systems, the host cells or host cell cultures which transfects with the vector systems just described are. Preferred hosts are unicellular prokaryotic organisms, especially E. coli. For expression of eukaryotic target genes can it is advantageous to use eukaryotic expression systems to z. Eukaryotic-typical posttranslational modifications in to introduce the target gene product. Particularly suitable eukaryotic host cells are yeasts, such. As the genus Aspergillus, Schwanniomyces, Kluyveromyces, Yarrowia, Arxula, Saccharomyces, Schizosaccharomyces, Hansenula, Pichia, Hanseniaspora, Zygosaccharomyces, Ustilago, Debaryomyces, Cryptococcus, Rhodotorula, Trichosporon, Kluyveromyces, Torulopsis or Williopsis.

With Help of the expression systems according to the invention succeeds, by coexpression of the target gene with the reporter gene, the activity of the target gene product in vivo. So z. B. by in vivo Expression of a hydrolase and the pHluorin determines the hydrolase activity be because the expressed hydrolase by the enzymatic reaction their substrates changed the pH in the host cell. This is all the more surprising because the pH in a cell is regulated by the cell.

So succeeds z. B. the esterase from Pseudomonas fluorescens in common to express with pHluorin. The esterase from Pseudomonas fluorescens is an enzyme that contains a number of aliphatic and aromatic esters in a temperature range up to 40 ° C to hydrolyze and is active in the pH range of 5 to 10.

For the coexpression of the esterase and the pHluorin reporter protein in E. coli, an expression vector pGEX_pH_PFE was prepared, which carries both the PFE gene of the PFE esterase and the pHluorin gene in each case under the control of its own promoter. This was constructed from plasmids pJOE 2792.1 and pGEX_rat_pHluorin. The cloning strategy is in 2 shown. For this purpose, a HindIII restriction cleavage site was first introduced by site-directed mutagenesis at the 3 'end of rat_pHluorin in the plasmid pGEX_rat_pHluorin. In the second step, the gene for the esterase pfe with its own promoter rhaP was cloned via the restriction sites EcoRI and HindIII from the plasmid pJOE2792.1 into the modified plasmid pGEX_rat_pHluorin.

By Co-expression of the PFE and pHluorin genes may alter the pH in the transformed host cell evoked by the expressed Hydrolase, be detected by means of the pHluorin reporter.

Consequently Another object of the present invention is a process for in vivo detection of the influence of the target gene products on the pH value in the host organism, whereby enzymes can be found or the enzymatic activity the enzymes can be determined.

• a) Coexpression des Zielgens und des Reportergens im Wirtsorganismus und
• b) Bestimmung der pH-Wert-Änderungen im Wirtsorganismus, die durch das Zielgenprodukt hervorgerufen wird, mit Hilfe des Reportergenproduktes.

The claimed method comprises at least the following method steps:

• a) Coexpression of the target gene and the reporter gene in the host organism and
• b) Determination of the pH changes in the host organism, which is caused by the target gene product, using the reporter gene product.

The Host cells can in a for the appropriate host organism suitable culture medium are grown. Cultivation can be achieved by plating the host cells on agar plates under selection of the expression systems with the addition of an antibiotic respectively. Alternatively, the cultivation of the host cells in liquid Culture medium possible, whereby selection by means of an antibiotic is advantageous, but not mandatory. The cultivation of the cells in liquid culture can be done in a comprehensive all expression systems container or separately in individual compartments, e.g. B. in a microtiter plate.

to Cultivation of expression systems, such. B. transfected with the vector systems of the invention E. coli strains, can the known culture media are used. For E. coli cultivation is z. For example, the Luria-Bertani Medium common. Antibody resistance genes are mostly used as selection markers, already in the common ones Vectors are integrated. For selection then the culture media appropriate antibiotics such as ampicillin, streptomycin, etc. added.

The Cultivation of the host cells is carried out according to standard protocols preferred at temperatures between 30 and 45 ° C.

In a preferred embodiment are used for the coexpression of target and reporter gene in the expression system Used vectors whose transcription is inducible. Thereby For example, the expression of the target and / or the reporter gene can be determined via the Addition of an inductor to be controlled. In this regard has become z. B. the induction by rhamnose and IPTG in conjunction with the Transcription control of target and reporter gene by the rha promoter and the tac promoter prove suitable.

The Efficiency of the described method can be further increased, by the medium for the respective enzyme suitable substrates are added, whereby the significance of the enzymatically induced pH change elevated.

In a further preferred embodiment is with the method according to the invention the catalytic activity of enzymes that catalyze pH-dependent reactions. This is the activity from enzymes over the strenght the pH change determined in the host cell.

With Help of this procedure it is possible above all the specific activity of individual Enzymes opposite to determine a particular substrate. In this way, for the organic Synthesis, based on given substrates, particularly efficient Enzymes are detected and thus provided. So z. B. to study the activity esterases are added as a substrate to the medium ester, the be enzymatically cleaved under release of protons.

at the implementation of certain substrates, it may be appropriate to transform the host cells into a suitable medium. The used for it Media preferably contain a buffer, with a pH range adjusted between pH 4 and pH 11, preferably between pH 5 and pH 10 can be. As a medium for ester cleavage in E. coli z. B. a Tris buffer (pH 7 - 9) be used. In this case, the selected buffer on the respective examined enzyme can be tuned. Also the buffer concentration can for the particular assay can be varied and optimized. So have yourself z. As in the study of hydrolases, in particular of esterases Proven phosphate buffer, in a concentration of over 50 mM, preferably between 100 and 1000 mM, more preferably between 250 and 750 mM.

Farther It may be advantageous to the culture solution, in addition to the respective Substrate also a solvent mediator, such as B. gum arabic or dimethyl sulfoxide (DMSO), to add.

The evaluation of the activity of the target gene products of individual transfected host cells or host cell colonies can, when using pHluorin as a reporter, z. B. by the spectrometric determination of individual, grown on an agar plate or in a microtiter plate expression cultures. The pH value change in the host cell is determined by determining the intensity _{ratio of} the emission _bands R _485/390 by means of a fluorimeter.

One another big one Advantage of a spectrometric pH determination is that it also transformed the search of whole libraries from Host cells allowed for a pH change in vivo. there may be the in vivo expression of target and reporter gene in liquid culture medium done, the culture solution can then z. By means of FACS (Fluorescence Activated Cell Sorting) simply analyzed and the host cells that have a strong enzymatic activity have isolated and thus be selected.

Moreover, the method described represents an important component, namely the selection step, for the generation of tailor-made enzymes by means of artificial protein evolution. The generation of artificial target gene mutants can be carried out, for example, by B. by mutagenizing the expression systems z. B. by UV radiation, radioactive radiation or DNA damaging substances. However, preference is given to the generation of mutant gene forms by the amplification of specific target genes with an increased error rate. These are z. B. used in the PCR amplification of the target genes inaccurately working DNA polymerases. The set of artificial target gene forms thus generated can then be converted back into the above-described vek be installed gates. After transfection of the host cells with these vectors, the target genes are expressed and selected again by measuring the pH change. The evolutionary cycle can be repeated several times.

One Another object of the present invention are gene banks containing the vector systems according to the invention or the expression systems of the invention. These include, in particular, libraries involved in the implementation of the Evolution cycles are generated.

Short description of Characters:

1 Figure 2 shows the expression plasmid pJOE 2792.1 for the expression of the esterase from Pseudomonas fluorescens (PFE) under the control of the rhamnose _inducible promoter P _rha and pGEX_rat_pHluorin for the expression of the GST-pHluorin fusion protein under the control of the P _tac promoter.

2 shows the cloning strategy for the construction of the coexpression plasmid pGEX_pH_PFE.

3 the graphical representation of the results of coexpression of pHluorin and PFE in E. coli DH5α and JM109.

4 shows SDS gels for controlling the enzyme expression of Bacillus stearothermophilus esterase (BSE) in E. coli.

5 Figure 9 is a graph showing the results of the coexpression of pHluorine and the Bacillus stearothermophilus esterase (BSE) in E. coli DH5α.

6 shows the influence of different solubilizers on the coexpression results of pHluorin and BSE.

7 shows the comparison of the emission ratios between E. coli cells transfected with the wild-type esterase from Bacillus stearothermophilus and with an inactive mutant of this esterase.

8th shows the comparison of the emission ratios between E. coli cells transfected with the wild-type esterase from Pseudomonas fluorescens (PFE) and with an inactive mutant of this esterase.

9 shows the results of a pHluorin assay in microtiter plate format.

10 shows the results of a kinetics measurement using the pHluorin assay.

11 shows a FACS analysis of E. coli DH5α cells.

12 shows a FACS analysis of E. coli DH5α cells with pHluorin activity

13 Figure 2 shows a FACS analysis of E. coli DH5α cells with pHluorin and esterase activity.

14 shows a FACS analysis of E. coli DH5α cells with pHluorine and an esterase at a phosphate buffer concentration of 50 mM, 15 shows a corresponding analysis at a buffer concentration of 500 mM.

The 16 to 18 showed a FACS analysis of E. coli DH5α cells with pHluorine and an esterase in a medium at pH 8.0, pH 7.0 and pH 6.

EXAMPLES

Example 1: pHluorin assay for determining the enzymatic activity of the Pseudomonas fluorescens esterase (PFE)

General

The following embodiment shows a fluorescence-based in vivo assay for the hydrolysis activity of Pseudomonas fluorescens esterase (PFE).

The Pseudomonas fluorescens esterase is a hydrolase that has a number aliphatic and aromatic esters in a temperature range up to 40 ° C to hydrolyze and active in a pH range of 5 to 10 is.

The released in the hydrolase-catalyzed substrate hydrolysis protons provide for a lowering of the pH. In order to be able to detect this pH change and thus also the hydrolase activity in vivo, the ratiometric pHluorin is also expressed in a cell in addition to the hydrolaseease to be investigated. Depending on the strength of the pH change by the PFE substrate conversion, there is a change in the two pH-dependent maxima in the excitation spectrum of the pH, which is detectable with the aid of a fluorescence spectrometer. The change in the emission _ratio R _475/395 from both emission _peaks is a measure of the pH change in the cell.

Step 1: In vivo Screening System for the hydrolysis activity of esterases

Construction of the vector pGEX_pH_PFE

The PFE gene was amplified by PCR from plasmid pJOE 2792.1 (Pelletier, I. and Altenbuchner, J. (1995); A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity., Microbiology 141, 459-68) the primer
PFE_Ecol: 5'-GGA GAT ATA CAT GAA TTC AGC ACA TTT GTT GC-3 '(Seq ID No. 1) and
PFE_HindIII: 5'-CCA AAA CAG AAG CTT GGC TGC-3 '(SEQ ID NO: 2)
amplified.

For the co-expression of the esterase and the pHluorin as reporter protein in E. coli, an expression vector pGEX_pH_PFE was prepared, which carries both the PFE gene of the PFE esterase and the pHluorin gene in each case under the control of its own promoter. For this purpose, a HindIII restriction cleavage site was first introduced by site-directed mutagenesis at the 3 'end of rat_pHluorin in the plasmid pGEX_rat_pHluorin. In the second step, the gene for the esterase pfe with its own promoter rhaP was cloned via the restriction sites EcoRI and HindIII from the plasmid pJOE2792.1 into the modified plasmid pGEX_rat_pHluorin ( 2 ). The resulting plasmid is referred to as pGEX_pH_PFE.

Mutagenesis was performed with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and PCR. For this purpose, the following primers were used:
MUT_pGEX1: 5'-GAA CTA TAC AAA TAA TGA GAA TTC ATC GTG AAG CTT TGA CGA TCT GCC TCG-3 '(Seq ID No. 3)
MUT_pGEX2: 5'-CGA GGC AGA TCG TCA AAG CTT CAC GAT GAA TCC TCA TTA TTT GTA TAG TTC-3 '(Seq ID No. 4)

To carry out the PCR, the pGEX_pH_PFE vector was suspended in 10 μl of 10 × Pfu buffer. After addition of 5 pmol (2.5 μl) each of the primers Seq. ID No. 3 and 4 were pipetted into 2.0 μl of a dNTP mixture (2.5 mM each) and 1.0 μl of a PfuTurbo polymerase solution (2.5U / 1 μl). The reaction mixture was made up to 100 ml with water. The PCR cycles were carried out with the following temperature program:
Program step 1: 95 ° C, 4 min
Program step 2: 95 ° C, 1 min; 48 ° C, 2 minutes; 72 ° C, 10 minutes; Repeat 25 times
Program step 3: 72 ° C, 10 min

After the reaction was digested with 2 ul Dpnl for 1 hour at 37 ° C. The mixture was then dialyzed against dH ₂ O for 2 hours. Thereafter, the transformation was carried out in E. coli DH5α. Served as an assay solution a solution of 2% gum arabic and 2% ethyl butyrate in Luria Bertani medium (LB medium).

Step 2: Production competent cells and their transformation.

The Transformation of the expression host is carried out according to the PEG / DMSO method (Chung, C.T., Niemela, S.L. and Miller, R.H. (1989); One-step preparation of competent Escherichia coli: transformation and storage of bacterial Cells in the same solution. Proc Natl Acad Sci U S A 86, 2172-2175).

For this purpose, 500 .mu.l of an overnight culture of E. coli DH5.alpha. Were inoculated into 50 ml of LB medium and incubated for 2 to 4 hours at 37.degree. C. in a shaking incubator to an optical density of OD ₅₇₈ = 0.4 to 0.7. The cells were centrifuged in a Hettich centrifuge at 3000 rpm for 10 min at 4 ° C. All further steps were also carried out at 4 ° C. After draining the supernatant, the cell pellet was resuspended in 2 ml of ice-cold TSS. 200 .mu.l of the cells were either used directly after 20 minutes incubation on ice for transformation or flash frozen in liquid nitrogen and stored at - 80 ° C.

Transformation of competent cells:
In each case 200 μl of the competent cells, 10 μl of ligation mixture or 1 μl of the plasmid DNA to be transformed were pipetted, briefly mixed and incubated on ice for 30 min. Subsequently, the transformation mixture was exposed for 30-45 sec to a heat shock of 42 ° C. To regenerate the cells, 0.8 ml of LB or SOC medium was added and incubated for one hour at 37.degree. Thereafter, the cells were centrifuged off at 7500 rpm for 2 min and re-suspended in the return. The cell suspension was plated on LB agar plates with appropriate selection antibiotic and incubated overnight at 37 ° C.

Step 3: Coexpression of pHluorin and PFE as well as detection of esterase activity in vivo in E. coli DH5α

For this purpose, pGEX_pH_PFE were transformed into E. coli DH5α. These cells are incubated at 37 ° C. in Luria-Bertani medium supplemented with ampicillin (100 μg / ml) until the exponential phase (OD ₆₀₀ 0.5-0.6, approximately 3 hours) is reached.

Subsequently, by addition of rhamnose (final concentration 0.2% w / v) and IPTG (final concentration 1 mM), co-expressed with pHluorin and PFE. 4 h after induction of the esterase and pHluorin, the cells are transferred into 50 mM Tris pH 8 with or without ethyl butyrate (final concentration 5% (w / v)) and incubated at 30 ° C. for 30 min. Subsequently, R _{485/390 was determined} in the fluorimeter (Fluorstar from BMG Labtechnologies, Offenburg).

Coexpression of PFE and pHluorin in E. coli DH5α with addition of ethyl butyrate as substrate reveals that the R _485/390 ratio is at a value of 2. The reference value of the R _485/390 ratio of the control _experiment without substrate is set at 1. The results are in 3 shown schematically.

coexpression of pHluorin and PFE as well as detection of esterase activity in vivo E. coli J M109

For this purpose, pGEX_pH_PFE were transformed into E. coli JM109. These cells are incubated at 37 ° C. in Luria-Bertani medium supplemented with ampicillin (100 μg / ml) until the exponential phase (OD ₆₀₀ 0.5-0.6, approximately 3 hours) is reached.

Subsequently, by addition of rhamnose (final concentration 0.2% w / v) and IPTG (final concentration 1 mM), co-expressed with pHluorin and PFE. 4 h after induction of the esterase and pHluorin, the cells are transferred into 50 mM Tris pH 8 with or without ethyl butyrate (final concentration 5% (w / v)) and incubated at 30 ° C. for 30 min. Subsequently, R _{485/390 was determined} in the fluorimeter.

In the coexpression of PFE and pHluorin in E. coli JM109 with the addition of ethyl butyrate as substrate even an R _485/390 ratio of 3.2 compared to the control _experiment without substrate (R _485/390 ratio of the control _experiment receives the value 1) receive. The results of the expression experiments are schematic tables in 3 played.

In Both expression systems may decrease the pH due to the expression of the hydrolase in vivo with the help of the pHluorine clearly be detected. The detection of the catalytic activity of the esterase is thus easily feasible with the present assay.

Protein evolution:

Based on that in the pGEX_pH_PFE vector, a library of mutant PFE genes can be generated by PCR. These can z. For example, the primers with Seq. ID. No. 3 and Seq. ID. No. 4 are used. To carry out the PCR, the pGEX_pH_PFE vector is suspended in 10 μl of 10 × Pfu buffer. After addition of 5 pmol (2.5 μl) each of the primers Seq. ID No. 3 and 4, 2.0 μl of a dNTP mixture (2.5 mM each) and 1.0 μl of a polymerase solution (2.5 U / 1 μl) are pipetted in. The reaction mixture is made up to 100 ml with water. The PCR cycles are carried out with the following temperature program:
Program step 1: 95 ° C, 4 min
Program step 2: 95 ° C, 1 min; 48 ° C, 2 minutes; 72 ° C, 10 minutes; Repeat 25 times
Program step 3: 72 ° C, 10 min

After the reaction was digested with 2 ul Dpnl for 1 hour at 37 ° C. The mixture was then dialyzed against dH ₂ O for 2 hours. Thereafter, the transformation takes place in E. coli DH5α. The library of artificially altered PFE genes thus generated can be re-assayed using the method described in step 3. The assay solution used is a solution of 2% gum arabic and 2% ethyl butyrate in LB.

Example 2: pHluorine Assay to determine the activity the esterase from Bacillus stearothermophilus (BSE)

a) Analogously to Example 1 were E. coli DH5alpha cells with a Bacillus esterase gene stearothermophilus (BSE) and the pHluroin gene.

The Esterase was from the strain Bacillus stearothermophilus IFO12550 isolated and fully characterized (Kugimiya, W., Ph. D. Thesis 1988, Kyushu University, Fukuoka, Japan). The pH range from 6.0 to 8.5, in which the BSE shows activity, is a little smaller than the PFE. It shows those for esterases typical substrate specificity for short-chain fatty acids such as 4-nitrophenylacetate and tributyrin.

b) Control of co-expression of pHluorine and the esterase from Bacillus stearothermophilus SDS-PAGE

to Control of coexpression of the fusion protein GST-pHluorin and Esterase from Bacillus stearothermophilus was each a cell culture prepared in the GST-pHluorin (reference cells) or GST-pHluorin and esterase was actively expressed. After induction was hourly Milliliter removed and over SDS-PAGE analyzed.

4 shows the SDS gels of reference (left) and esterase active cells (right). In 4 In the case of the reference (left side) as well as the cells with esterase activity (right side), a clear overexpression of the GST-pHluorin fusion protein at 56.5 kDa is seen after four hours. In addition, the esterase-active cells show a band at 30.6 kDa, which corresponds to the molecular weight of the Bacillus stearothermophilus esterase.

c) assay establishment for various substrates

Application of the pHluorin Assay on the esterase from Bacillus stearothermophilus with various substrates

E. coli DH5α cells, which in addition to pHluorin also have esterase activity, were resuspended in potassium phosphate buffer (pH 8.0) with 2% substrate and 2% gum arabic. After incubation at 37 ° C. for 40 min, 250 μl thereof were transferred to black microtiter plates, the fluorescence at the two excitation wavelengths 390 nm or 485 nm was measured and the emission _ratio R _485/390 as described in Example 1 was determined incubated for four hours at 37 ° C, the cells were spun down and resuspended in potassium phosphate buffer (pH 8.0) containing 2% substrate (m / v). For enzyme catalytic hydrolysis, the cells are incubated for a further 40 minutes at 37 ° C. Subsequently, R _{485/390 was determined} in the fluorimeter. The reference used was the determination of the emission _ratio R _485/390 of E. coli DH5α cells in which pHluorin, but not the esterase was expressed.

In 5 the results obtained are shown. The investigated esterase shows an extremely low activity compared to butyl acetate and tricaprylin, which is also reflected in the small changes in the emission ratios compared to the reference cells without esterase activity. With sales of substrates such as tributyrin and phenylacetate, for which the esterase has a high activity, almost a doubling of the emission ratio can be seen.

d) Influence of the phosphate buffer concentration on the fluorescence activity of pHluorine

The influence of the phosphate buffer concentration on the fluorescence activity was investigated with phenylacetate as substrate. For this E. coli DH5α cells, which in addition to pHluorin and esterase activity (BSE) possess in different potassium phosphate buffer concentrations (pH 8.0) with 2% phenyl acetate and 2% gum arabic resuspended. After 40 min incubation at 37 ° C., 250 μl thereof were transferred to black microtiter plates, the fluorescence at the two excitation wavelengths 390 nm and 485 nm, respectively, and the emission _ratio R _{485/390 determined} as just described. In turn, the emission _ratio R _485/390 of E. coli DH cells served as a reference in which pHluorin, but not the esterase was expressed. The results of the measurements are shown in Table 1 below.

Tab. 1: Comparison of buffer concentration and fluorescence intensities of pHluorin

e) Influence of the solubilizer on the pHluorin assay

For the pHluorin assay, various solubilizers and their influence on the fluorescence activity of pHluorin and the emission _ratio R _{485/390 were} investigated. For this purpose, as described in Example 2 E. coli DH5α cells, which in addition to pHluorin also esterase activity (BSE) possess in potassium phosphate buffer (pH 8.0) with 2% substrate (tributyrin, phenylacetate) and various solubilizers resuspended. After 40 min incubation at 37 ° C., 250 μl thereof were transferred to black microtiter plates, the fluorescence at the two excitation wavelengths 390 nm and 485 nm, respectively, and the emission _ratio R _485/390 determined as described above. In turn, the emission _ratio R _485/390 of E. coli DH5α cells served as a reference in which pHluorin, but not the esterase was expressed.

The use of gum arabic compared to dimethyl sulfoxide and Tween 20 for both substrates shows the largest change in the emission ratio ( 6 ). However, investigations on the FACS have shown that E. coli attaches to the gum arabic particles finely distributed in the emulsion, thus making the analysis and sorting of the cells more difficult (data not listed). With regard to a successful transfer of the pHluorin assay to the FACS, dimethylsulfoxide has proven to be a better alternative.

In 6 the comparison of the emission ratios when using different solubilizers is shown graphically.

Example 3: Comparison the esterase activity of the wild type with the esterase activity of a mutant using the pHluorin assay

It investigations were carried out whether the pHluorin assay for the screening of mutants can be used. For this purpose were by means of site-specific mutagenesis inactive mutants of the esterases from Bacillus stearothermophilus and Pseudomonas fluorescens Exchange of active serine for alanine and prepared in E. coli XL1-Blue transformed.

The Coexpression of pHluorin and inactive mutants or pHluorin and Wild type in E. coli DH5α cells was carried out as described in Example 1. Subsequently were the Activities mutant and wild-type with the pHluorin assay using of phenylacetate as a substrate. In turn served as a reference E. coli DH5α cells, in which only pHluorin was expressed.

The results are in 7 shown. 7 shows the comparison of emission ratios between cells with esterase activity and with an inactive mutant esterase from Bacillus stearothermophilus.

8th shows the comparison of emission ratios between cells with esterase activity and cells with an inactive mutant esterase (PFE) from Pseudomonas fluorescens.

The application of the pHluorin assay to the deactivated mutant esterase from Bacillus stearothermophilus with phenylacetate as substrate shows, as expected, no change in the emission ratio compared to the reference cells ( 7 ). The same applies to the esterase from Pseudomonas fluorescens ( 8th ). However, the emission ratio of the inactive mutant is slightly smaller (~ 1.10) than that of Bacillus stearothermophilus esterase.

Example 4: Establishment of the pHluorin assay for Screeening in microtiter plate format

a) Use of the pHluorin Assay in microtiter plate format for screening for esterase activity of Bacillus esterase stearothermophilus (BSE)

Eight individual colonies transfected with the wild-type gene or the inactive mutated gene variant of the esterase from Bacillus stearothermophilus were cultured in microtiter plates (96-well plates from Greiner). Subsequently, the induction of pHluorin and esterase with IPTG and rhamnose solution in the microtiter plates, whereby the final concentrations described in Example 1 were achieved. After four hours of incubation at 37 ° C, the cells were transferred into potassium phosphate buffer (pH 8.0) with phenyl acetate and gum arabic and incubated for an additional 40 minutes at 37 ° C. Subsequently, the fluorescence was measured at the two excitation wavelengths 390 nm and 485 nm and the emission _ratio R _485/390 determined (analogous to Ex. 1). Eight different batches of E. coli DH5α cells in which only pHluorin was expressed were also used as reference.

The results are in 9 shown that the emission ratio of the cells expressing the active wild-type enzyme show emission ratios between 2 and 2.5, which are in a similar range as in Schüttelkolbenansatz (see Example 2). The same applies to cells expressing the inactive mutated enzyme variant and for cells without esterase activity (reference), which emission conditions in the range from 0.9 to 1.3. The emission ratios of these cells is thus clearly below the cells expressing active esterases. It could thus be shown that cells cultivated directly in microtiter plate format with and without esterase activity can be distinguished directly in the microtiter plate with the aid of the pHluorin assay.

b) Kinetic uptake of pHluorine assays with substrate conversion in microtiter plate format

Four single colonies with the esterase from Bacillus stearothermophilus were cultured in microtiter plates. Subsequently, the induction of pHluorin and esterase with IPTG and rhamnose solution and another four hours expression time at 37 ° C. To _monitor the change in emission _ratio R _485/390 with decrease in pH during substrate _hydrolysis , the cells were transferred to potassium phosphate buffer (pH 8) with 2% phenyl acetate and 2% gum arabic and incubated for an additional 40 minutes at 37 ° C , The fluorescence was measured every minute at the two excitation wavelengths 390 nm and 485 nm, and the emission _ratio R _{485/390 was} determined. Four different batches of E. coli DH5α cells, in which only pHluorin was expressed, also served as a reference.

The results are in 10 shown. 10 shows the change in the emission ratio of pHluorin in the hydrolysis of phenyl acetate as a function of the reaction time. While the emission ratio of the E. coli cells without esterase activity (lower four curves) remains almost constant during the hydrolysis reaction of phenylacetate starting from a value of about 1.3, in cells with esterase activity (upper four curves) a marked increase of the emission ratio. After about 35 to 40 minutes. the substrate is completely reacted. As a result, there is no longer any change in pH, and thus a constant value for the emission ratio also sets in here. At the beginning of the hydrolysis reaction, the cells already have an R _485/390 value of 1.7, which increases to 2.5 to 3.2 in the course of the reaction.

Example 5: Identification and separation of cells with esterase activity using the pHluorine assay at the FACS

Around the possibility of the pHluorin Assay for Single Cell Sorting Using Esterase from Bacillus stearothermophilus at FACS were studied as in Example 2 described various approaches of cells and cultured the expression induced by the addition of IPTG and rhamnose. To 4 hours, the cells were as usual transferred in potassium phosphate buffer with or without substrate and 3% DMSO as a solubilizer and the substrate hydrolysis by incubation for 40 min. forced at 37 ° C. Subsequently were the cell solutions measured at the FACSScan.

In the following examples, pHluorine absorption was performed at 475 nm using a blue laser (488 nm, FL1 filter) and a second laser (390 nm, FL2 filter). The flow cytometers FACSScan ^™ and FACSVantage ^™ with FACSDiVa ^™ option from Becton-Dickinson were used.

a) Measurement of unlabelled E. coli DH5α cells

First, measurements were carried out with unlabelled cells, which serve as comparison to pHluorin-active cells ( 11 ).

As expected, virtually all detected cells show both a weak FL1 and FL2 signal. The defined region R1 contains 96.80% of all cells (events). The region R1 is characteristic of colorless E. coli DH5α cells, without esterase activity. The measurement results are in 11 and listed in the following table:

b) Measurement of E. coli DH5a cells with pHluorin activity after substrate conversion

For comparison with cells in which, in addition to pHluorin, esterase activity is also present, E. coli DH5α pGEX_rat_pHluorin cells were measured with tributyrin as the substrate in which pHluorin was previously expressed ( 12 ).

In region R2 are 77.10% with a geometric fluorescence mean of 217.58 for the FL1 signal, in R1 only 17.60% (geometric mean of 3.49). Consequently, cells with pHluorin activity show a significantly higher FL1 signal than colorless E. coli. The region R2 is characteristic of pHluorine-labeled cells. The measurement results are in 12 and shown in the following table.

c) Measurement of E. coli DH5a cells with pHluorin and esterase activity after substrate turnover

Before measurement, pHluorine and the esterase from Bacillus stearothermophilus were first expressed and the cells were then transferred into 50 mM potassium phosphate buffer (pH 8) with tributyrin and dimethyl sulfoxide (DMSO) and incubated for 40 min at 37 ° C. The results are in 13 and shown in the following table.

The Evaluation of the results shows that the cells with esterase and pHluorin activity a weaker FL1 signal across from Show cells with pHluorin but without esterase activity. This is also evident in the geometric mean of the fluorescence signal of 132.80. in the Compared to colorless E. coli show the pHluorin / esterase-labeled Cells significantly higher Fluorescence values. The R3 region, which accounts for 74.00% of all cells (Events) is characteristic of pHluorine-labeled cells with esterase activity.

d) Comparison of E. coli DH5a cells with pHluorin and esterase activity when reacting different Substrates at different buffer concentrations

In front the measurement was first pHluorin and the esterase from Bacillus stearothermophilus expressed and then the cells in 50 mM or 500 mM potassium phosphate buffer (pH 8) with tributyrin and dimethylsulfoxide and incubated for 40 min at 37 ° C.

At a buffer concentration of 50 mM, the FL1 signal of the cells with pHluorin and esterase activity is almost completely lost after substrate conversion with phenyl acetate ( 14 ). Almost all cells (events) can be found in region R1 (97.60%). The cells can no longer be distinguished from colorless E. coli DH5α. This result also correlates with the values measured in the fluorescence spectrometer in Example 2, according to which the cells almost completely lose their fluorescence signal at a buffer concentration of 50 mM after conversion with phenyl acetate. The results of the measurement are shown in the following table:

At a buffer concentration of 500 mM, however, a clear FL1 signal was obtained ( 15 ). In the region R2 characterized by pHluorine-labeled cells, there are 76.90% of all cells (events). The cells are clearly distinguishable from colorless E. coli. The results of the measurement are shown in the following table:

e) Comparison of cells with pHluorin and esterase activity at different pH's

Around a change to detect fluorescence as the pH dropped, cells became with pHluorin and esterase activity in 50 mM potassium phosphate buffer resuspended at different pH and after incubation for 40 min. at 37 ° C examined at the FACS.

For the characterization of the cells (events) the region R1 was defined ( 16 . 17 . 18 ). At pH 8.0 in R1 the cells show a geometric mean for FL1 of 183.93 ( 16 ). As the pH decreases, the geometric mean increases above 232.03 at pH 7.0 ( 17 ) up to 264.78 at pH 6.0 ( 18 ). This showed the increase in the FL1 signal of the cells as the pH dropped. The results of the measurements are shown in the following tables:

By the studies on FACS showed that pHluorine-labeled cells without or with esterase activity can be distinguished from colorless E. coli. In addition, in cells with pHluorin activity, the FL1 signal increases at decreasing pH, which is in direct correlation to the excitation spectrum of pHluorin, its maximum at 475 nm with decrease in pH gets bigger. It is important, however, that at all pH values a pH change through the esterase activity can be determined in vivo.

SEQUENCE LISTING

Claims (22)

Vector system comprising at least one vector containing at least one transcribable from a promoter Target gene and at least one, of the same or of another Promoter from transcribable pH-sensitive reporter gene. Vector system according to claim 1, characterized in that at least one target gene codes for an enzyme, that is a pH dependent Catalyzed reaction. Vector system according to claim 2, characterized in that the target genes independently of one another for an enzyme selected from the group of oxidoreductases, transferases, hydrolases or Lyases encoded. Vector system according to one the claims 1 to 3, characterized in that the vector system of at least two vectors, wherein at least one vector is a pH-sensitive Contains reporter gene and at least one other vector contains a target gene. Vector system according to one the claims 1 to 4, characterized in that the vector system of at least a vector consisting of at least one target gene and at least a pH-sensitive reporter gene. Vector system according to one the claims 1 to 5, characterized in that the pH-sensitive reporter gene the pHluorin gene or a variant thereof, with a sequence homology of at least 50%. Vector system according to one the claims 1 to 6, characterized in that the vector or the vectors of the vector system containing transcriptional regulatory sequences. Vector system according to claim 7, characterized in that the vector system is the pGEX_rat_pHluorin Vector contains. Vector system according to one the claims 1 to 8, characterized in that the vector system has a Containing target gene containing pJOE vector. Vector system according to one of the claims 1 to 7, characterized in that the vector system consists of a pGEX_pH_Zielgen coexpression plasmid exists. Expression system comprising one with a vector system according to claims 1 to 10 transfected host cell or host cell culture. Expression system according to claim 11, characterized in that that the host cells are unicellular pro- or eukaryotic organisms are. Expression systems according to one of claims 11 or 12, characterized in that the Host cell is an E. coli cell or a yeast cell. Compound library containing vector systems according to one the claims 1 to 10. Expression library containing expression systems according to one the claims 11 to 13. Use of the vector systems according to one of claims 1 to 10 for transfecting cells or cell cultures. Use of the expression systems according to the claims 11 to 13 for Method for finding pH-dependent reactions catalyzing Enzymes or to determine the catalytic activity of such Enzymes. Method for finding pH-influencing Target gene products from a host cell containing a vector system according to claims 1 to 10 comprising the following method steps: a) Coexpression of the Target gene and the reporter gene in the host organism and b) determination the pH changes in the host organism evoked by the target gene product, with the help of the reporter gene product. Method for finding pH-influencing Target gene products according to claim 18, characterized in that the coexpression of the target gene and / or of the reporter gene is induced. Method according to claim 18 or 19, wherein for identification of an enzyme of interest an expression system according to the claims 11 to 13 or an expression library according to claim 15 containing pHluorin as reporter gene spectrometrically at a wavelength of 450 nm to 500 nm and / or 360 nm to 440 nm are investigated. Method according to the previous Claim, characterized in that the emission ratio of both bands is determined spectrometrically. Method according to one the claims 18 to 21, characterized in that the expression of the target genes of expression systems in liquid Medium with subsequent Separation of positive host cells by means of FACS takes place.