WO2002064773A2 - Procede de selection a haut rendement de composes candidats par synthese directe a haut rendement de polypeptides recombinants - Google Patents

Procede de selection a haut rendement de composes candidats par synthese directe a haut rendement de polypeptides recombinants Download PDF

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WO2002064773A2
WO2002064773A2 PCT/DE2002/000459 DE0200459W WO02064773A2 WO 2002064773 A2 WO2002064773 A2 WO 2002064773A2 DE 0200459 W DE0200459 W DE 0200459W WO 02064773 A2 WO02064773 A2 WO 02064773A2
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sequence
dna
tag
protein
high throughput
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PCT/DE2002/000459
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WO2002064773A3 (fr
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Hans-Joachim Müller
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Mueller Hans-Joachim
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present connection relates to a method for high-throughput direct synthesis of recombinant polypeptides which, after coupling to a carrier matrix, can be used directly for high-throughput screening of candidate compounds.
  • the method according to the invention permits the in vitro synthesis of more than 5,000 recombinant Proteins per day.
  • HTE High Throughput Expression
  • the interaction between proteins and their potential binding partners can be carried out fully automatically in a high throughput expression process ("High Throughput Expression” (HTE)) with subsequent high throughput screening.
  • HTE High Throughput Expression
  • the necessary gene segments are reversely transcribed and amplified via (RT-) PCR.
  • the derived recombinant proteins are then expressed in vitro by the method according to the invention and fixed on a carrier matrix (for example biochips): tedious and time-consuming cloning is eliminated.
  • a carrier matrix for example biochips
  • the bound proteins are exposed to potential binding partners (pharmaceuticals, etc.) and their affinity for one another is then measured.
  • the free interaction partners can be labeled with a fluorescent dye, for example, which ensures a reliable statement about the binding affinity of both partners by fully automated spectroscopic methods.
  • biochips can also be used repeatedly for drug target screening.
  • the present invention thus relates to a method for the high-throughput direct synthesis of recombinant polypeptides and their coupling to a carrier matrix, which is characterized by the following steps: (a) Preparation of DNA constructs which have the following sections:
  • Partial sequences e.g. exon regions of genomic DNA
  • the specific gene sequences are duplicated using suitable molecular biological methods (e.g. PCR, RT-PCR etc.). These DNA amplificates are then rewritten into RNA using RNA polymerases and then translated into polypeptides by in vitro translation. The polypeptides obtained can then be used for interaction or activity analyzes.
  • genomic DNA A specific genomic sequence consisting of an entire operon, different exon / intron sequences or individual exon regions (eg exons containing SNP ("single nucleotide polymorphism")) is used (eg as a data file ) determined. According to the wishes / requirements, the specific gene sequences are amplified and used for the method according to the invention.
  • the necessary and species-specific template (genomic DNA) can also be provided by the client as well as by other sources.
  • cDNA and mRNA The sequence data and possibly the template (eg PCR fragments, vectors, syn- Theoretical genes etc.) with details of the gene segment to be processed by the client.
  • the template is in turn amplified via PCR and used for the method according to the invention. If, for example, only the cDNA sequence is communicated, then the required cDNA can be produced by isolating and reverse transcribing species-specific mRNA using RT-PCR.
  • Protein sequences Starting from an isolated and possibly provided protein, its N-terminal protein sequence is determined by sequencing and degenerate oligonucleotides are derived and synthesized from the protein sequence obtained. This, in turn, for example, be used in combination with poly (A) 15 _ 20 oligonucleotides for RT-PCR from the required biological starting materials (eg. As a piece of fabric) was used. If the protein sequences can be provided, the protein sequencing is omitted. After receiving the gene-specific amplificate, the protein sequence derived therefrom is verified by DNA sequencing. General methods known in the art can be used to prepare the DNA constructs of the invention.
  • the method according to the invention for high-throughput direct synthesis of recombinant proteins is further characterized in that the DNA constructs additionally have the following sections: a multiple cloning site located 5 'to the promoter sequence, one between the coding for an affinity tag DNA sequence and the DNA sequence encoding the desired recombinant polypeptide, an amino acid linker encoding sequence, and / or a 3 'to the polypeptide encoding DNA sequence, a poly (dA) encoding DNA sequence, the length of the poly (dA) tail encoded by this DNA sequence is preferably 15 to 50 dA, and the DNA sequence preferably also has a multiple cloning site.
  • a multiple cloning site located 5 'to the promoter sequence, one between the coding for an affinity tag DNA sequence and the DNA sequence encoding the desired recombinant polypeptide, an amino acid linker encoding sequence, and / or a 3 'to the polypeptide encoding DNA sequence
  • the amino acid linker preferably consists of 5 to 20 glycine and / or serine residues.
  • the DNA constructs according to the invention are preferably produced by PCR, with e.g. can be carried out according to the schemes described in Example 1 below.
  • the DNA constructs are particularly preferably produced using megaprime PCR, which, for example, in the following examples from Barik and Galinsky, BioTechniques 10. (1991), 489-490.
  • Any promoter which leads to strong gene expression in the transcription / translation system used is suitable for the transcription of the DNA constructs used in the method according to the invention.
  • Suitable promoters are known to the person skilled in the art.
  • the T7, T3 or SP6 promoter is particularly preferred, although other promoters such as the E.coli S30, the SV40 or the HCMV promoter can also be used.
  • Affinity tags suitable for the method according to the invention and the corresponding binding partners as well as conditions for the covalent or non-covalent coupling of the binding partner to a carrier matrix are known to the person skilled in the art.
  • suitable affinity systems are the Strep-Tag-System, (Institute for Bioanalytics, Göttingen, Germany), or the T7-Tag-System, (Novagen, Madison, USA), which are a Strep-Tag affinity protein or binding partner contain a T7 tag antibody.
  • the S-peptide / S-protein system described in the examples below is preferably used for the process according to the invention.
  • the present invention further relates to a method for high throughput screening, which is characterized by the following steps: (a) contacting those according to that described above Procedure obtained polypeptides bound to a support matrix with one or more candidate compounds; and (b) determining the binding of a candidate compound to a polypeptide.
  • the candidate compounds can be very different compounds, both naturally occurring and synthetic, organic and inorganic compounds, as well as polymers (eg oligopeptides, polypeptides, oligonucleotides and polynucleotides) as well as small molecules, antibodies, sugars, Fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (eg peptide "imitators", nucleic acid analogs etc.) and numerous other compounds.
  • polymers eg oligopeptides, polypeptides, oligonucleotides and polynucleotides
  • small molecules antibodies, sugars, Fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (eg peptide "imitators", nucleic acid analogs etc.) and numerous other compounds.
  • the search for suitable compounds can also be carried out on a large scale, for example by screening a very large number of candidate compounds in substance libraries, where the substance libraries can contain synthetic or natural molecules.
  • the candidate compound is part of a substance library.
  • a large number of potentially useful molecules can be screened in a single test. For example, if a field of 1000 compounds is to be screened, in principle all 1000 compounds can be placed in a microtiter plate well and tested at the same time. If a binding is discovered, the pool of 1000 can then be divided into 10 pools of 100 and the process repeated until an individual connection is identified. is adorned. In any event, the production and simultaneous screening of large banks of synthetic molecules can be carried out using well known combinatorial chemistry techniques, see for example van Breemen, Anal. Chem. 69. (1997), 2159-2164 and Lam, Anticancer Drug Des. 12: 145-167 (1997).
  • extracts from natural products as a starting material can come from a large number of sources, for example the species fungi, actinomycetes, algae, insects, protozoa, plants and bacteria.
  • the extracts that indicate binding can then be analyzed to isolate the active molecule. See, for example, Turner, J. Ethnopharmacol. 51 (1-3) (1996), 39-43 and Suh, Anticancer Res. 15 (1995) 233-239.
  • Preferred candidate compounds are SMDs, peptides, hormones, proteins and nucleic acids.
  • the candidate compounds preferably carry a detectable label, which is preferably a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate or an enzyme.
  • a detectable label which is preferably a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate or an enzyme.
  • the person skilled in the art is familiar with methods for detecting whether the candidate compound has bound to the recombinant polypeptide as a function of the label chosen for the candidate compound.
  • Preferred methods for determining the binding of a candidate compound are radioactive, fluorescence-dependent, magnetic, spectroscopic, mass spectrometric, biosensory, photometric methods or confocal laser microscopy. Conditions for carrying out these processes are known to the person skilled in the art.
  • Suitable carrier matrices are also known to the person skilled in the art, a microtiter plate, a glass slide, a reaction vessel, a membrane, a biochip (The Chipping Forecast 1999, Nature Genetics 21: Supplement) or a gel matrix are preferred as carrier matrices.
  • Gel matrices eg gel filtration or ion exchange matrices
  • suitable gel matrices are streptavidin-coupled column materials (eg Sephadex TM, DE ⁇ E etc., Pharmacia, Freiburg, Germany), for example for the binding of biotinylated S protein (Novagen, Madison, USA).
  • FIG. 1 Amplification of the specific gene or ORF
  • fragment A Schematic representation of the gene-specific PCR fragment (fragment A). Fragment A is extended by the two oligonucleotides by approximately 80 to 100 nucleotides. The area marked in bold represents the gene-specific sequence of the oligonucleotides.
  • FIG. 1 Schematic representation of the gene-specific 5'-01igonucleotide (A).
  • the gene-specific area is italicized and bold.
  • the white arrow indicates the direction of synthesis.
  • the initiation signal "ATG" of the specific gene is in the ORF of the upstream linker sequence.
  • (C) Schematic representation of the gene-specific 3'-01igonucleotide (A). The gene-specific area is italicized and bold. The sequence of this 3-oligonucleotide must be shown as a complementary strand. The gray arrow indicates the direction of synthesis. The stop codon is underlined. A poly (A) tail of 15 adenines is inserted as a translation-promoting sequence.
  • the length of the fragment is approximately 90 nucleotides. Hatched rich: multiple cloning site; gray area: promoter sequence for RNA polymerases; black area: S-peptide sequence; white area: complementary linker sequence.
  • Promoter sequence is underlined.
  • the white arrow indicates the direction of synthesis.
  • (C) Schematic representation of the 3 'oligonucleotide (B) of fragment B.
  • the 5' S peptide region is marked in bold.
  • the sequence of this 3 'oligonucleotide must be shown as a complementary strand.
  • the gray arrow indicates the direction of synthesis.
  • Figure 3 Schematic representation of hybridization between the ssDNA 3 'region of fragment B and the ssDNA 5' region of fragment A
  • the polymerase extends the 3 'ends in the direction of the fragment B and fragment A region (gray arrows).
  • MCS multiple cloning site
  • Prom RNA promoter
  • SPS S-peptide sequence
  • GSS-T gene-specific sequence with poly (dA) tail.
  • FIG. 4 Schematic representation of the megaprime product.
  • the length of the megaprime product depends on the length of the gene-specific fragment (GSS).
  • GSS gene-specific fragment
  • the length of the upstream and downstream DNA sequences is uniform.
  • MCS multiple cloning site;
  • Prom RNA promoter;
  • SPS S-peptide sequence;
  • Gly (n) glycine linker sequence;
  • poly (dA) ls _ 50 poly (dA) tail.
  • the length of the total protein depends on the length of the gene-specific protein sequence (GSPS).
  • S-peptide S-peptide sequence
  • Gly (n) glycine linker sequence
  • ⁇ H 2 amino-terminal protein end
  • COOH carboxy-terminal protein end.
  • the linker between S-protein (hatched) and carrier matrix is marked as a gray wave.
  • the S protein bound to the carrier matrix black bar
  • GSP recombinant protein
  • Example 1 Preparation of the DNA constructs that can be used for the method according to the invention via PCR (a) Amplification of the specific gene
  • fragment A 1 (FIG. 1 a ).
  • the 5 'oligonucleotide (A) used for the preparation of fragment A has the gene-specific sequence at the 3' end (15-20 nucleotides for the zinc finger gene pATl33 (Müller et al., PNAS USA 88. (1991), 10079- 10083; 5 'ATG CTC CAC CTT AGC-3') (FIG. 1b).
  • a "linker sequence" of 12-24 bp (eg 5 '-GGG GGG GGG GGG GGG-3') is added upstream of this sequence, which results in a sequence of 4-8 glycine side chains after translation
  • the 3 'oligonucleotide (A) used for the preparation of fragment A likewise has the gene-specific sequence with 15 to 25 nucleotides at its 3' end (the stop codon End of the pAT133 gene containing CTA: 5 '-CTA TTC ACT GGG TGG-3'), the 5 'end of the 3' oligonucleotide (A) additionally being a sequence of translation-promoting sequences (for example poly (A) 10 _ 50 ) includes ( Figure lc).
  • thermostable DNA polymerase (Hybaid GmbH, Heidelberg, Germany), (2 U / ⁇ l), lOx reaction buffer complete (200mM Tris-HCC1, pH 8.5, 160mM (NH 4 ) 2 S0 4 , 15mM MgCl 2 ), 5 'Oligonucleotide (A) (50 pmol / ⁇ l), 3' oligonucleotide (A) (50 pmol / ⁇ l), dNTP mix (40 mM), gene-specific template (total amount 0.1-10 ng); e.g. pATl33 plasmid, H 2 0 bidist., sterile reaction vessels (0.5 ml / 0.2 ml)
  • PCR fragment B Amplification of the PCR fragment B
  • a further "PCR fragment B" is amplified, which contains the sequences shown in FIG. 2a.
  • the 5′-ligonucleotide used for the PCR is composed of three sequence elements (FIG. 2b): At the 5 ′ end, various interfaces for restriction enzymes are contained (eg 5 ′ -GGA TCC GAA TTC CCC GGG-3 ′; for Ba HI , EcoRI, Smal etc.).
  • a promoter sequence of strong viral, bacterial or eukaryotic RNA polymerase is inserted downstream thereof (15-30 nucleotides: eg T7 RNA pol promoter: 5'-ATA TAACACAC TCA CTA TAGGGC GA-3 ') and on 3' - The end of this 5 'oligonucleotide (B) closes the 5' sequence for the S peptide of bovine RNase A from the bovine pancreas (Richards and Wyckoff, The Enzymes Vol. IV (1971), 647-806, Academic Press) with 21 nucleotides (e.g. 5 'ATG GAA ACT GCA GCA GCC AAG-3').
  • the ATG initiation signal opens the ORF of the entire fusion product.
  • the 3 'oligonucleotide (B) for the PCR of the fragment B has at its 5' end the complementary sequence (5 '-CCC CCC CCC CCC-3') of the linker sequence of the gene-specific 5 'oligonucleotide (see figure lb) on.
  • the last 15-30 nucleotides e.g. 5 '-GTC CAT GAG CTG CCG CTC AAA CTT-3'
  • Reaction conditions for the amplification of the q-specific fragment B materials thermostable DNA polymerase (2 U / ⁇ l), lOx reaction buffer complete, 5 'oligonucleotide (B) (50 pmol / ⁇ l), 3' oligonucleotide (B) (50 pmol / ⁇ l), dNTP mix (40 mM), RNase A template (Schmidt et al., Biochemistry 25 (20), 1986, 5955-5961; 1245825 (GenBank)) (total amount 1 ng / ⁇ ), H 2 0 bidist., Sterile reaction tubes (0.5 ml / 0.2 ml)
  • Execution (pipetting scheme for 50 ⁇ l final volume): add 50 ⁇ l H 2 0 bidist .: 5.0 ⁇ l lOx reaction buffer complete, 1.0 ⁇ l 5 'oligonucleotide (B), 1.0 ⁇ l 3' oligonucleotide (B) , 1 ul RNase A template, 2.0 ul dNTP mix, 0.5 ul thermostable DNA polymerase
  • RNA or mRNA must be used as the starting material for the gene-specific fragment A, then the oligonucleotides (A) are used in the RT-PCR as follows.
  • RT-PCR program / fragment A 1st step: 6 min 70 ° C, 2nd step: 5 min / 48 or 58 ° C (depending on the 3 'oligonucleotide), 3rd step: 30 min / 60 ° C, 4th step 2 min / 94 ° C, 5.
  • the PCR fragments are purified using standard methods (silica beads, spin preps) in accordance with the manufacturer's instructions.
  • the purified fragments A and B are then used for the megaprime PCR.
  • the so-called megaprime PCR (Barik and Galinsky, BioTechniques 10. (1991), 489-490) allows overlapping DNA fragments to be ligated together directly during the PCR cycles.
  • the denatured single DNA strands serve as primers and bind the complementary sequence of the overlap partner.
  • the 3 'oligonucleotide (B) and the 5' oligonucleotide (A) have a complementary sequence ("linker sequence"), so that after denaturation of the two fragments, hybridization between the complementary ends of the fragments A and B can take place ,
  • the hybridized single strands can now act as "megaprimer" so that the polymerase elongates the free 3 'ends of these strands (FIG. 3).
  • both the 5'-oligonucleotide (B) and the 3'-oligonucleotide (A) are pipetted into the PCR mixture.
  • Execution (pipetting scheme for 100 ul final volume): 10 ul 10 x reaction buffer complete, 76 ul H 2 0 bidist., 2.0 ul 5 'oligonucleotide B, 3.0 ul 3' oligonucleotide A, 3.0 ul Fragment A, 3.0 ul fragment B, 2.0 ul dNTP mix, 1.0 ul Taq / Pwo DNA polymerase mix.
  • the fragment obtained can be used directly for the combined in vitro transcription / translation.
  • Example 2 Cell-free in vitro transcription / translation
  • Example 1 The fragment produced in Example 1 is used for this.
  • A the eukaryotic (reticulocyte lysate or wheat germ lysate) or bacterial "TNT TM Coupled" transcription / translation systems from Promega, Mannheim, Germany are used.
  • Example 1 "TNT-Coupled” transcription / translation system, TNT reaction buffer, TNT RNA polymerase, amino acid mixture including [ 35 S] labeled amino acids (Promega, Mannheim, Germany), RNAsin inhibitor (Promega, Mannheim, Germany) (40 U / ⁇ l), H 2 0 bidist. (DEPC-treated, nuclease-free).
  • the procedure is adapted to the volumes required for the process according to the invention (approx. 5-20 ⁇ l) in accordance with the manufacturer's instructions.
  • Pipetting scheme for 10 ul final volume in "384well" microtiter plates xx ul TNT lysate, 0.4 ul TNT reaction buffer, 0.2 ul TNT RNA polymerase, 0.2 ul amino acid mixture, 0.8 ul [ 35 S] - labeled Amino acid (1000 Ci / mmol, lOmCi / ml), 0.2 ⁇ l RNasin inhibitor, xx ⁇ l megaprime PCR fragment (0.1 to 0.5 ⁇ g), add 10.0 ⁇ l H 2 0 bidest.
  • the individual components (apart from the Megaprime PCR fragment) are pipetted as a master mix and placed in the corresponding microtiter wells.
  • the Megaprime PCR fragment is added directly to the microtiter wells.
  • the reaction is carried out at 30 ° C for 30-120 min.
  • Example 3 Binding of the recombinant proteins via the affinity tag to a carrier matrix
  • the proteins obtained from Example 2 are then bound to the required carrier matrices.
  • Appropriately pretreated materials can be used as the carrier matrix, preferably "384well” polystyrene or polypropylene microtiter plates or glass slides being used.
  • the S protein is non-covalently bound to the carrier matrix to be used, (a) microtiter plates and (b) glass slide carriers being used.
  • the recombinant proteins are then coupled via the S-peptide to the carriers pretreated in this way.
  • Couple proteins and e.g. for high-throughput screening with the addition of potential interaction partners SMDs
  • the recombinant proteins obtained in Example 2 (5-20 ⁇ l translation batch) are pipetted directly into the corresponding microtiter wells of the prepared microtiter plates without prior purification.
  • the recombinant proteins (0.2 - 1.0 ⁇ l translation batch) are pipetted directly into the corresponding microtiter wells of the prepared microtiter plates without prior purification.
  • Example 4 Interaction analysis of the proteins coupled to the carrier After being fixed to the carrier matrices (Example 3), the proteins translated in vitro were used to screen potential interaction partners.
  • the variability in the screening system according to the invention is given by the fact that a large number of different proteins or mutants can be applied per glass slide surface / microtiter plate and that the slide is incubated with only one molecule to be examined, for example in a slide wash bowl (FIG. 7A ).
  • various molecules to be examined can be transferred to the different protein spots / wells of the glass slides or microtiter wells using pipetting or spotting robots (FIG. 7B).
  • the zinc finger protein of the pATl33 gene and its binding partner (dsDNA sequence. 5 '-GGGGCGGGG-3') was used as an example for an interaction analysis between a protein which was translated in vitro according to Example 2 and locally fixed according to Example 3 and a free labeled ligand.
  • dsDNA sequence. 5 '-GGGGCGGGG-3' was used as an example for an interaction analysis between a protein which was translated in vitro according to Example 2 and locally fixed according to Example 3 and a free labeled ligand.
  • Gel retardation Gibresl. Vol. 2, Greene Publ. Assoc., Inc. J. Wiley & Sons, NY , USA
  • footprint analyzes Schomitz and Galsa, Nucl. Acids. Res. 6 (1978), 111).
  • the master mixes (Ix gel shift binding buffer) prepared, then pipetted 10 ul master mix per well of the "384well" plate.
  • unlabeled Competitor DNA was pipetted in a five-fold molar excess to the expected amount of protein, after which it was incubated at room temperature for 5-10 min.
  • a five-fold molar excess was added to the expected amount of protein Cy3-labeled dsDNA and it was incubated for 5-10 min at room temperature.
  • the supernatant was removed and washed twice with 1 x gel shift binding buffer. Subsequently, the whole was left in 10 ⁇ l of 1 x gel shift binding buffer, evaluated with the aid of a microtiter plate fluorescence reader, and the binding between the zinc finger protein and its binding partner was detected.
  • the proteins in vitro translated according to Example 2 and bound to the carrier matrix according to Example 3 can also be used to find potential medicaments for or for certain proteins in the high-throughput method.
  • Human phenylalanine hydroxylase which has a natural affinity for L-phenylalanine, was used.
  • SNP point mutation
  • L-DOPA L-DOPA
  • the interaction between L Phenylalanine and the human phenylalanine hydroxylase have been demonstrated several times with in vitro translated proteins (Knappsog et al., Hum.
  • the slide was preincubated in PBS and then the excess liquid was removed by draining and wiping gently. This was followed by transferring 0.2 ⁇ l (for “384well” plates) or 1.0 ⁇ l (for “96well” plates) of the L-phenylalanine solution (50 ⁇ M-500 ⁇ M) to a corresponding slide spot. The incubation was carried out for 30 min at room temperature. The slide was then washed twice in PBS. Finally, the excess liquid was removed by draining and wiping gently. Bound L-phenylalanine molecules were analyzed by glass slide fluorescence readers (chip leader, Virtek Inc., Waterloo, Canada).

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Abstract

L'invention concerne un procédé de synthèse directe à haut rendement de polypeptides recombinants qui, après couplage à une matrice support, peuvent être utilisés directement pour une sélection à haut rendement de composés candidats.
PCT/DE2002/000459 2001-02-09 2002-02-07 Procede de selection a haut rendement de composes candidats par synthese directe a haut rendement de polypeptides recombinants WO2002064773A2 (fr)

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