WO2004036205A2 - Kit de marquage de poids moleculaire pour proteines et procede pour le produire - Google Patents

Kit de marquage de poids moleculaire pour proteines et procede pour le produire Download PDF

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Publication number
WO2004036205A2
WO2004036205A2 PCT/EP2003/010548 EP0310548W WO2004036205A2 WO 2004036205 A2 WO2004036205 A2 WO 2004036205A2 EP 0310548 W EP0310548 W EP 0310548W WO 2004036205 A2 WO2004036205 A2 WO 2004036205A2
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Prior art keywords
molecular weight
weight marker
marker kit
protein
kit according
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PCT/EP2003/010548
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German (de)
English (en)
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WO2004036205A3 (fr
Inventor
Thomas Brodegger
Hartmut Follman
Wolfgang Nellen
Jürgen OBERSTRASS
Anja Stockmann
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Universität Kassel
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Publication of WO2004036205A2 publication Critical patent/WO2004036205A2/fr
Publication of WO2004036205A3 publication Critical patent/WO2004036205A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • the present invention relates to a molecular weight marker kit which is used to analyze proteins on the basis of their different rates of migration in the electric field.
  • the invention further discloses a method for producing a molecular weight marker kit.
  • the principle of separation is based essentially on the fact that almost all biological molecules, such as amino acids, peptides, proteins and nucleic acids, carry ionizable groups and can therefore be present in solution as electrically charged compounds, either as cations (+) or anions (-).
  • electrically charged compounds either as cations (+) or anions (-).
  • the differences in the charge densities are evident in different migration speeds in the electric field.
  • the rate of migration depends on the force of the electric field on the ions and the frictional and electrostatic see resistance between the connections and the surrounding milieu.
  • the sample to be separated must be dissolved in a buffer solution for electrophoresis, and all carrier materials must be soaked with buffer solution in order to transmit the current.
  • All carrier materials suitable for electrophoresis have a capillary network, which are common to those skilled in the art: filter papers, cellulose acetate, thin layers of silica gel or aluminum oxide, starch, agar or polyacrylamide gels.
  • a matrix made of polyacrylamide gel is particularly suitable for the separation of proteins with good selectivity.
  • the preparation of a polyacrylamide gel from the monomer acrylamide and the cross-linker N, N "-methylene (bisacylamide) in the presence of a catalyst is known to the person skilled in the art.
  • the percentage of acrylamide is determined the pore size of the gel, gels between 3 and 30% acrylamide have a pore size of 0.5 nm and 0.2 nm diameter. 30% gels are used for the separation of proteins with a molecular weight of up to 10 4 daltons (Da), for the separation of proteins up to 10 6 Da 3% gels. Gradient gels are suitable for the particular selectivity of proteins with very large differences in the molecular masses since they contain a concentration gradient of acrylamide and thus a change in the pore size within the gel. SDS polyacrylamide gel electrophoresis is frequently used in protein analysis in a discontinuous polyacrylamide gel developed according to Laemmli (Nature, 1970).
  • a form of this method of polyacrylamide gel electrophoresis modified according to Schwarz and Jagow is particularly suitable for analyzing small proteins with 5-20 kDa, since the tricine used instead of glycine enables a sharper separation.
  • the gel is clamped in an electrophoresis apparatus with an upper and lower buffer reservoir, the sample with the proteins to be separated is applied to the wells of the gel and exposed to a voltage for a certain time in which the proteins are removed from the sample according to the described
  • the different calibration proteins of the standard marker mixture have the same or at least a similar running behavior in the gel as the proteins of the sample to be determined, an estimate or a determination of the molecular masses of the protein in the sample is possible via a calibration curve.
  • the migration distances are inversely proportional to the log TM of the molecular mass.
  • the calibration proteins of the standard marker mixture each have different running behavior in the gel due to their size and shape and are no longer suitable for creating an exact calibration curve, since the migration distances are no longer inversely proportional to the log 10 of the molecular mass are.
  • EP 0518476 A1 discloses a method for protein determination in capillary electrophoresis, wherein two external markers with known running properties are added to the sample to be examined. A method for identifying band positions in an electrophoretic separation of a sample on a gel using an internal standard is described in DE 691 27 999 T2.
  • the standard markers thus contain proteins of defined sizes, but the running properties of the individual proteins during electrophoresis are influenced differently by other criteria that do not depend on the size - such as the steric conformation and shape of the proteins.
  • the friction and electrostatic interactions attack differently with each marker protein, so that the migration distances are no longer inversely proportional to the log TM of the molecular mass.
  • the molecular mass of a protein to be investigated cannot be read with sufficient accuracy from a standard curve created therefrom.
  • the invention is therefore based on the central object of providing a molecular weight marker kit which is more suitable for analyzing proteins on the basis of their different migration speeds in the electric field and avoids the disadvantages of the prior art described.
  • the invention provides special molecular complexes or protein complexes which are more suitable with regard to their migration speeds in the electrical field, since secondary influences have no effects.
  • the standard markers according to the invention (or the different molecular weights) are generated in that several identical molecules are combined so that the different molecular weights can be set.
  • the object is achieved according to the invention with a molecular weight marker kit for analyzing proteins on the basis of their different rate of migration in the electric field, characterized in that a) the molecular weight marker kit essentially consists of a single protein component and homomultimers in that b) the size of the multimers Has multiple times the number of a monomer, and that c) the homomultimers are present under oxidative conditions.
  • the present invention further provides a method for producing a molecular weight marker kit, which comprises the following steps:
  • the process can be carried out very simply and inexpensively, since only a single protein component has to be produced instead of 5-8 different components and their mixture in a suitable ratio.
  • simple oxidative conditions are required to use the molecular weight marker kit for multimerization of the protein.
  • FIG. 2 shows a schematic representation of the mutation of the DdTRXI protein by site-directed mutagenesis
  • FIG. 3 a schematic representation of the plasmid pGEM3ZDdTRX1.
  • the term multimer means protein complexes composed of two to at least ten peptides which are assembled and which are present as homodi, tri, quattro, penta- and at least homo-decamers.
  • Thioredoxins are ubiquitous regulatory proteins of every pro- and eucaryotic cell, they are small, soluble, heat-stable proteins with a molecular weight of 12 kDa consisting of 105-110 amino acids. Its active center is highly conserved and consists of a characteristic tetrapeptide Cys-Gly-Pro-Cys, often flanked by trypthophane at the N-terminus and a basic amino acid at the C-terminus.
  • DdTRXI Dictyostelium discoideum
  • the two cysteines contained can act as an H donor or acceptor by reversibly changing between the oxidized disulfide and the reduced dithiol form.
  • the mutation according to the invention is a mono-serine-thioredoxin
  • the mono-serine-thioredoxin mutant DdTRXI-C35S contains further sequence differences, for example so-called conservative substitutions, in which amino acids from the same class are exchanged for one another. This means that, for example, an amino acid with a polar side chain is replaced by another amino acid with an equally polar side chain. Possible sequence differences are also insertion (s) or deletion (s), provided that they do not concern the active center.
  • a sequence homology of at least 70% will preferably and particularly preferably a sequence homology of 80% to the sequence of the mono-serine-thioredoxin mutant DdTRX1-C35S.
  • the mono-serine-thioredoxin mutant DdTRXI-C35S shows, under simple oxidizing conditions and by adding oxidizing agents, for example in the presence of 10 mM diamide, multimerization of the monomer to di-, tri, quattro-, penta- to at least Decamer, where the molecular weight is a multiple of the monomer (13.8 kDa; theoretically calculated using a computer program; for practical applications, the molecular weight of the monomer is rounded to 14 kDa).
  • oxidizing agents for example in the presence of 10 mM diamide, multimerization of the monomer to di-, tri, quattro-, penta- to at least Decamer, where the molecular weight is a multiple of the monomer (13.8 kDa; theoretically calculated using a computer program; for practical applications, the molecular weight of the monomer is rounded to 14 kDa).
  • the mono-serine-thioredoxin mutant DdTRXI-C35S according to the invention is therefore suitable as a molecular weight marker kit for analyzing proteins on the basis of their different rate of migration in the electric field. Since these are homomultimers, the friction and electrostatic interactions attack in the same way, so that the migration distances are exactly inversely proportional to the log-io of the molecular mass. On the one hand, this molecular weight marker kit is very well suited for estimating the molecular mass of an unknown protein sample, and on the other hand, by creating a standard curve, the molecular mass of the protein to be examined can be read very precisely.
  • each band of the molecular weight marker kit according to the invention can also be transferred to commercially available membranes in the further biochemical analyzes (for example Western blot).
  • the molecular weight marker kit is particularly well suited for the analysis of bio-tiny proteins in a Western blot. Detection on the membrane is carried out using special antibodies or more simply using streptavidin-alkaline phosphatase (or alternatively streptactin
  • FIG. 1 shows the molecular weight marker kit A according to the invention in comparison to a conventional molecular weight marker kit B.
  • the separation of the regions with the molecular weight marker kit A and that with the molecular weight marker kit B is identified by a dashed line 4.
  • the proteins are applied to a polyacrylamide matrix under non-reducing conditions (without the addition of DTT) and separated according to their size in an electrical field (from cathode ⁇ to anode +) and then transferred to a nitrocellulose membrane and by means of streptactin phosphatase ® (IBA, Göttingen) colored.
  • the direction of migration in the electric field is shown by an arrow 2.
  • Each band represents a protein of a certain size, which is given on each side in the SI unit for proteins in kDalton.
  • the molecular weight marker kit used for comparison contains seven different molecular weights, which thus results in seven different bands 10.
  • the bands 10 represent molecular weights from 6 kDa to 105 kDa.
  • the molecular weight marker kit according to the invention comprises at least 10 different molecular weights which range from approximately 14 kDa to at least 138 kDa.
  • This molecular weight marker kit thus contains at least ten different bands 20 which, in the range from approximately 14 kDa to at least 138 kDa, the monomer, the di-, tri, quattro-, penta- to at least decamer form of the mono-serine-thioredoxin Mutant DdTRXI- Show C35S. It goes without saying that a smaller or larger number of molecular weight differences can also be selected by a user.
  • the size of the monomer can be expanded as desired. For example, fusion of the mutated TRX gene with the open reading frame of another gene that is as inert as possible produces a larger monomer, which is also multimerized by the TRX portion.
  • a molecular weight marker kit that extends to at least 600 kDa is produced from a 60 kDa monomer, for example. Such a molecular weight marker kit is also used in gel filtration.
  • the present invention further provides a method for producing the molecular weight marker kit according to the invention, which comprises the following steps:
  • oligonucleotide-controlled mutagenesis as is achieved with site-directed mutagenesis (site-specific mutagenesis)
  • site-directed mutagenesis site-specific mutagenesis
  • kits are available to the person skilled in the art for carrying out them (for example QuickChange®, site-directed mutagenesis kit from Stratagene). 2 shows in schematic form the steps for introducing a mutation into the DdTRXI protein by site-directed mutagenesis.
  • Starting material forms a plasmid 12 with the wild-type sequence of the DdTrxI thioredoxin gene and specific oligonucleotides (mutagene se primers 1 and 2) which contain the desired mutation (here: TGT GGC CCA TCT) and flanking nucleotides of the wild-type sequence.
  • 3 shows a schematic representation of the plasmid pGEM 3Z-DdTrx1 used as starting material for the site-directed mutagenesis.
  • the wild-type DdTrx1 gene is cloned into plasmid pGEM-3Z® (Promega) via the restriction sites Sac I and Sa HI.
  • Each plasmid (pGEM 3Z-DdTrx1) 12 contains the target sequence to be mutated 14.
  • the mutagenic oligonucleotides hybridize with the wild-type DNA, and DNA strands with a correctly base-paired mutation site are formed in the PCR reaction.
  • the DNA double strand is then cut from contained or introduced specific recognition sequences for restriction endonucleases, incorporated into a suitable expression vector, transformed into a host cell and the protein overexpressed under appropriate induction conditions.
  • Expression vectors are also widely described in the literature.
  • they contain a promoter and usually a termination signal for the transcription.
  • At least one restriction interface or a polylinker is located between the promoter and the termination signal, which enable the insertion of a coding DNA sequence.
  • Promoters which can be used are those which bring about constitutive gene expression and also inducible ones which enable targeted gene expression.
  • the T7 promoter (Studier et al., 1990) for example, allows a very strong expression of the downstream gene. This leads to a high yield of protein.
  • the molecular weight marker kit according to the invention is particularly easily and quickly isolated if a modification is applied to the protein overexpression (method according to commercially available modification kits) and the protein is isolated directly by affinity chromatography according to the manufacturer's instructions. This takes place, for example, in the form of a Strep-Tag® (IBA GmbH, Göttingen), the detection of the proteins in the electrophoresis gel using conventional staining methods, in the vest nblot via commercially available streptavidin-alkaline phosphatase, Strep-Tactin-Phosphatase® (IBA GmbH, Göttingen) or via special antibodies.
  • Strep-Tag® IBA GmbH, Göttingen
  • the host cells are cultivated in culture media that are adapted to the needs of the host cells in terms of pH, temperature, salt concentration, antibiotics, vitamins, trace elements and aeration.
  • Standard methods from the prior art are used to isolate the DdTRXI protein mutant from the host cell, for example isolation using affinity chromatography using StrepTag®.
  • the isolated protein is multimerized by oxidation, ie by adding an oxidizing agent, such as diamide, or by dispensing with the usual addition of a reducing agent, such as DTT.
  • oxidizing agent such as diamide
  • reducing agent such as DTT
  • Site-specific mutagenesis leads to the targeted creation of point mutations at a defined point in a gene.
  • the mutation is generated with the help of modified, synthetically produced oligonucleotides at the desired location, which serve as a primer 16 for a DNA polymerase.
  • the site-specific mutagenesis is carried out using a commercially available "QuickChange TM Site Directed Mutagenesis Kit” from Stratagene according to the manufacturer's instructions.
  • the starting construct and template is plasmid pGEM 3Z-Trx1 (the thioredoxin wild-type DNA sequence from Dictyostelium discoideum is cloned into the plasmid pGEM 3Z® available from Promega via the restriction sites Sacl and BamHI, described by T. Brodegger, molecular biological function analysis of thioredoxins Dictyostelium discoideum and identification of new interaction partners through the two-hybrid system, dissertation (2015) Kassel 2002) in supereoiled form from a standard plasmid maxipreparation (according to the manufacturer's instructions, Qiagen, Hilden), which the DdTrxI-
  • the primers 16 for the PCR reaction contain the point mutation C instead of wild type T, so that the amino acid 35 cysteine (codon TGT) is replaced by serine (codon TCT). In addition, this creates a new interface for the endonuclease Xba I, which is used for later identification of the mutated clones.
  • a silent mutation in amino acid 33 was also replaced to uniquely identify the plasmids containing the mutation against GGC, both code for glycine), which destroys an interface for the restriction enzyme Ava II.
  • the mutagenesis primers are exactly complementary to each other and have a Tm value of at least 78 ° C.
  • the mutagenesis position is underlined, the corresponding nucleotides are highlighted in bold letters.
  • the PCR reaction is carried out using commercially available buffer and polymerase under the following conditions:
  • the PCR mixture is mixed with 1-2 ⁇ l of Dpn I for 1.5 h at 37 ° C., so that the methylated original templates are removed by the Dpn I restriction endonuclease, so that only the DNA strands with the point mutation.
  • the DNA strands with the point mutation can be cloned by standard Proto ⁇ koll into a bacterial expression vector, for example the expression vector pET 15 b, which is under the control of the T7 promoter, in this case provided with the Strep-tag® sequence and Kochex ⁇ pression of the recombinant protein in an E. coli host strain trans ⁇ formed (e.g. BL21).
  • a bacterial expression vector for example the expression vector pET 15 b, which is under the control of the T7 promoter, in this case provided with the Strep-tag® sequence and Kochex ⁇ pression of the recombinant protein in an E. coli host strain trans ⁇ formed (e.g. BL21).
  • the overexpression takes place according to the manufacturer's instructions of the Novagen pET system in 100 to 500 ml bacterial cultures be shaken at 225 rpm and 30 ° C. Approximately 3 hours after inoculation with an overnight culture (1 ml per 50 ml culture), an optical density OD S oo of about 0.4 was reached in the bacterial culture.
  • Gene expression is induced by adding the galactoside of IPTG to a final concentration of 1 mM, which is recognized by the bacterial expression system as a lactose analogue and acts as an inducer for gene expression.
  • the cells are harvested by centrifugation in the cooling centrifuge for 15 min at 4000 rpm, the cell pellet is frozen at -80 ° C and stored until further use.
  • the overexpressed protein is purified using the commercially available S rep-tag® system, since the molecular weight marker kit according to the invention additionally carries a Streptag® and is isolated easily and quickly using affinity chromatography using a Strep-Tactin®-Sepharose® column according to the manufacturer's instructions becomes.
  • the starting material for cleaning is a cell pellet from 100 ml bacterial culture stored at - 80 ° C.
  • the cells When thawing and lysing the cells, they are resuspended in 1 ml buffer W (100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8.0), to which 5 mM DTT is added as a reducing agent.
  • the complete cell lysis and fragmentation of the nucleic acids is carried out using ultrasound. 20 ⁇ g of avidin is added to the cell extract in order to block bacterial biotinylated proteins such as BCCP (Biotin Carboxyl Carrier Protein) and to prevent their non-specific binding to the column material.
  • BCCP Biotin Carboxyl Carrier Protein
  • the mono-serine-thioredoxin mutant DdTRXI -C35S is contained in the 3rd to 5th elution fractions in high purity.
  • the monomer has a Mo ⁇ lekularthe of about 13.8 in the figure, the rounded value of 14 kDa is located.
  • an oxidizing agent for example diamide, is added to the protein preparation in a concentration of 1-15 mM, preferably 10 mM.
  • the modified form of the denaturing, discontinuous polyacrylamide gel electrophoresis according to Shugger and Jagow (1987) is particularly suitable for the analysis of small proteins with 5-20 kDa, since the tricine used instead of glycine enables a sharper separation.
  • the gels are analyzed, for example, as mini gels using a Hoefer apparatus and prepared according to the following instructions:
  • composition of the 10 °> i separating gel (amount for 2 mini gels):
  • composition of the bulk gel is Composition of the bulk gel:
  • the molecular weight marker kit and the protein sample to be analyzed are mixed with the appropriate amount of sample buffer (4-fold), mixed well and incubated at 40 ° C for 30-60 min for denaturation and possible reduction before the desired amount is applied to the gel ,
  • sample buffer does not contain a reducing agent such as DTT, since multimerization is reversible under reducing conditions.
  • a suitable amount of protein per sample is 10-50 ⁇ g.
  • the upper buffer reservoir is filled with cathode buffer, the lower buffer reservoir with anode buffer.
  • the gel run takes place at a constant 20 mA per mini gel for 2.5 to 3 hours.
  • the gel is then stained with Coomassie blue (or with another staining method) or subjected to any detection method.
  • the proteins are stained or detected (for example with phosphatase or antibodies) and by comparing the band positions of the molecular weight marker kit with the bands of the analytes proteins is the moles ⁇ külmasse the determined protein sample to be analyzed and a Calibration curve determined exactly.
  • the migration distances of the bands of the molecular weight marker kit are uniformly inversely proportional to the logio of the molecular mass.
  • All bands of the molecular weight marker kit are also transferred to commercially available membranes in further biochemical analyzes (such as Western blot) and can be detected via phosphatase and / or special antibodies (e.g. anti-StrepTag antibodies).
  • the size of the molecular weight marker kit can be expanded as desired by fusing the mutated TRX gene with the open reading frame of another gene that is as inert as possible, which also multimerizes through the TRX portion.
  • a 60 kDa monomer provides a molecular weight marker kit with bands up to at least 600 kDa.
  • Such a molecular weight marker kit is also used in gel filtration.

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Abstract

La présente invention concerne un kit de marquage de poids moléculaire qui sert à analyser des protéines grâce à leurs différentes vitesses de déplacement dans un champ électrique. Le kit de marquage de poids moléculaire est composé notamment principalement d'une composante protéique unique et d'homo-polymères de celle-ci, la taille des polymères comprenant un multiple du nombre de monomères, et la polymérisation s'effectuant dans des conditions d'oxydation. Le kit de marquage de poids moléculaire est produit grâce à un procédé simple et économique qui n'implique la production que d'une seule protéine.
PCT/EP2003/010548 2002-09-25 2003-09-23 Kit de marquage de poids moleculaire pour proteines et procede pour le produire WO2004036205A2 (fr)

Applications Claiming Priority (2)

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DE10244502.8 2002-09-25
DE2002144502 DE10244502B4 (de) 2002-09-25 2002-09-25 Molekulargewichtsmarker für Proteine und Verfahren zu dessen Herstellung

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WO2004036205A2 true WO2004036205A2 (fr) 2004-04-29
WO2004036205A3 WO2004036205A3 (fr) 2004-06-03

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Cited By (1)

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CN109668771A (zh) * 2019-02-26 2019-04-23 长沙协大生物科技有限公司 一种液基脱落细胞染色液及其染色方法

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WO2015025054A1 (fr) 2013-08-22 2015-02-26 Medizinische Universität Wien Anticorps spécifiques du colorant pour marqueurs de masse moléculaire pré-colorés et procédés de production associés
EP3227341A1 (fr) 2014-12-02 2017-10-11 CeMM - Forschungszentrum für Molekulare Medizin GmbH Anticorps anticalréticuline mutante et leur utilisation dans le diagnostic et la thérapie de tumeurs malignes myéloïdes

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US5449758A (en) * 1993-12-02 1995-09-12 Life Technologies, Inc. Protein size marker ladder
WO1998030684A1 (fr) * 1997-01-08 1998-07-16 Life Technologies, Inc. Procedes de production de proteines
WO2002048174A2 (fr) * 2000-12-15 2002-06-20 Stratagene Polypeptides fluorescents dimeres

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Title
SCHAEGGER H ET AL: "TRICINE-SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS FOR THE SEPARATION OF PROTEINS IN THE RANGE FROM 1 TO 100 KDA" ANALYTICAL BIOCHEMISTRY, ORLANDO, FL, US, Bd. 166, 1987, Seiten 368-379, XP000961523 ISSN: 0003-2697 in der Anmeldung erw{hnt *

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Publication number Priority date Publication date Assignee Title
CN109668771A (zh) * 2019-02-26 2019-04-23 长沙协大生物科技有限公司 一种液基脱落细胞染色液及其染色方法

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