WO2002029027A1 - Adn polymerase immobilisee - Google Patents

Adn polymerase immobilisee Download PDF

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Publication number
WO2002029027A1
WO2002029027A1 PCT/KR2001/001650 KR0101650W WO0229027A1 WO 2002029027 A1 WO2002029027 A1 WO 2002029027A1 KR 0101650 W KR0101650 W KR 0101650W WO 0229027 A1 WO0229027 A1 WO 0229027A1
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WIPO (PCT)
Prior art keywords
dna polymerase
immobilized
linker
immobilization
activity
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PCT/KR2001/001650
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English (en)
Inventor
Hyun Jin Hwang
Jeong Hee Kim
Original Assignee
Ahram Biosystems Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/KR2000/001104 external-priority patent/WO2002074993A1/fr
Priority claimed from PCT/KR2001/001239 external-priority patent/WO2003008570A1/fr
Application filed by Ahram Biosystems Inc. filed Critical Ahram Biosystems Inc.
Priority to AU2001294299A priority Critical patent/AU2001294299A1/en
Publication of WO2002029027A1 publication Critical patent/WO2002029027A1/fr
Priority to US10/406,154 priority patent/US7238505B2/en
Priority to US11/809,188 priority patent/US8067174B1/en

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent

Definitions

  • the present invention relates to a DNA polymerase immobilized by covalent bonding.
  • the present invention relates to an immobilized DNA polymerase which is immobilized by forming a covalent bond with at least one linker that is connected to a supporting material through chemical bonding; whose immobilization site where the linker will be connected is selected such that the DNA polymerase is oriented; and wherein the average number of the covalent bond per DNA polymerase is controlled, thereby achieving the average activity of the immobilized DNA polymerase more than 10% relative to that of the solution phase DNA polymerase.
  • Enzymes are proteins that catalyze chemical reactions in vivo, and they have very specific catalytic activities toward chemical reaction and substrate. Enzyme reactions using their specific catalytic activities are utilized in various researches and diagnosis in the life science and medical fields and also in various catalytic chemical processes to enhance their efficiencies. In these utilizations of enzyme reactions, it is required in many cases to separate enzymes from reaction solutions after the reactions are completed, for the purpose of purifying products or recycling the enzymes. However, in solution phase, it takes long time and is very cumbersome to separate and purify materials such as enzymes and reaction products from reaction samples. Therefore, it is very important in the utilization of enzyme reactions to develop a method of simplifying the enzyme separation process.
  • immobilized enzymes can provide a very efficient method to simplify the enzyme separation process, a variety of researches have been performed extensively on the development of methods for immobilizing enzymes.
  • the "immobilized enzyme” as used herein means an enzyme that is physically or chemically bound to a supporting material while retaining its catalytic activity. Advantages of using such immobilized enzymes are as follows. Firstly, the purification or separation process of reaction samples can be simplified since the immobilized enzyme can be easily separated and recycled from reaction samples by simply removing the immobilized enzyme after an enzyme reaction. Secondly, the cost can be reduced by reusing the recycled immobilized enzyme. Thirdly, because multiple processes where enzyme reactions are included can be simplified, the efficiency of the overall reaction processes can be increased. Lastly, the efficiency of utilizing enzyme can be increased due to additional effects such as an enhancement in physical stability of enzyme after immobilization or a change in the conditions of enzyme reaction.
  • Examples of the enzyme immobilization methods include the carrier-binding method where enzymes bind to a carrier that is insoluble to water, the cross-linking method where enzymes are connected one another using a reagent having multiple reaction groups, and the entrapping method where enzymes are surrounded by semi-permeable gel or macromolecular membrane.
  • the immobilized DNA polymerase in the present invention is prepared by a kind of the carrier-binding method where enzymes are immobilized on a supporting material with covalent bonding. The characteristics of the carrier-binding method are thus described in detail below.
  • the kind of a carrier and the binding method are selected based on physical and chemical interactions between the enzyme and the carrier. This is because the amount and the characteristics of the enzyme after immobilization on the carrier depend greatly on the characteristics of the carrier and the binding method.
  • This method is classified by the binding method between the enzyme and the carrier, and examples of this method include the physical adsorption method via hydrogen bonding and
  • the physical adsorption method or the ionic bonding method has an advantage that the possibility of damaging the enzyme activity by immobilization bonding is low, since the enzyme binding process to a carrier is relatively simple and the influence on the active site structure of the enzyme is relatively low due to weak binding.
  • the enzyme can be lost even with the small change in temperature and pH, due to the weak binding between the enzyme and the carrier, and the low specificity that causes unspecific binding of undesired proteins.
  • the enzyme Since the enzyme has various reaction groups that can form covalent bond with a carrier, for example, amine, carboxyl, hydroxyl, thiol, imidazole, etc., immobilization reactions toward such reaction groups that can be performed in aqueous solution have been developed such as amide bond formation reaction, alkylation or arylation, disulfide bond formation, diazotization.
  • immobilization reactions toward such reaction groups that can be performed in aqueous solution such as amide bond formation reaction, alkylation or arylation, disulfide bond formation, diazotization.
  • an immobilization method where an immobilization reaction group on a supporting material reacts directly with a reaction group of an enzyme, or an immobilization method where a supporting material and an enzyme are connected using a linker having reaction groups at both ends.
  • the covalent bonding method has a difficulty of preserving the enzyme activity after immobilization, since a strong bond formed between the enzyme and the carrier gives rise to the structural change in the enzyme which in turn increases the possibility of
  • the most important point in immobilizing an enzyme on a supporting material with covalent bonding is that damaging the enzyme activity due to a structural change in the enzyme active site by immobilization bonding must be prevented Therefore, it is essential that the immobilization reaction must not occur at or near the active site. And also, even if the immobilization reaction occurs as a site distant from the active site of the enzyme, the allosteric effect that eventually reduces the enzyme activity by influencing the total structure of the enzyme must not occur. Therefore, in order to immobilize the enzyme with its activity preserved, it must be possible to select an immobilization site in the enzyme toward which immobilization bonding occurs, i.e., to select a reaction group of the enzyme located at a particular site such that the activity is not damaged after the immobilization bonding.
  • an oriented immobilization should be possible wherein the enzyme can be immobilized as oriented so that a reaction group of the enzyme located at the particular site forms an immobilization bonding.
  • an enzyme is a macromolecule that has a great number of amino acids connected by amino bonding. Due to such characteristic of the enzyme, reaction groups available for immobilization reaction, for example, amine, carboxyl, and hydroxyls, are numerous and they are distributed throughout the enzyme. Therefore, it is impossible with the prior technologies developed to date to direct an oriented immobilization by selecting a reaction group of an enzyme located at a particular site. In other words, in the enzyme immobilization using the prior art immobilization methods, random immobilization reactions occur toward a plurality of reaction groups in the enzyme. Therefore, an immobilization bonding can be formed at an undesirable site or a plurality of immobilization bonding can be formed, thereby severely damaging the enzyme activity.
  • the formation of covalent bonding by itself can possibly reduce the enzyme activity largely because it requires rearrangement of electrons. That is, even if it is possible to select a reaction group of the enzyme located at a particular site so as to direct an oriented immobilization toward the reaction group, it is impossible to confirm presence of the covalently immobilized enzyme with its activity preserved without damage until an oriented immobilization is actually performed.
  • the DNA polymerase that was immobilized in the present invention is being utilized extensively in the nucleic acid amplification technology using polymerase chain reaction (PCR) and also in the DNA recombination processes in the field of genetic engineering for the purpose of research and development and clinical diagnosis.
  • PCR polymerase chain reaction
  • an immobilized DNA polymerase whose activity for DNA polymerization is preserved to be high enough to use in the DNA recombination processes and the PCR amplification technology has not been provided yet.
  • DNA polymerase whose average activity is preserved higher than 10% relative to that of a solution phase DNA polymerase. This will be achieved by making it possible that the immobilization site in the enzyme where a linker binds is selected such that the DNA polymerase is oriented for immobilization bonding, and also the average number of covalent bonding per immobilized DNA polymerase is controlled, while the DNA polymerase is immobilized via covalent bonding.
  • the present invention provides a novel immobilized DNA polymerase with highly preserved activity that is immobilized via covalent bonding.
  • the present invention provides an immobilized DNA polymerase consisting essentially of a DNA polymerase, a linker, and a supporting material, wherein the DNA polymerase forms covalent bonding with at least one linker; the linker forms chemical bonding with the supporting material; the immobilization site of the DNA polymerase that will be connected to the linker is selected such that the DNA polymerase is oriented; and the average number of covalent bonding per immobilized DNA polymerase is controlled, such that the average activity of the immobilized DNA polymerase is preserved more than 10% relative to that of the solution phase DNA polymerase.
  • the present invention further provides an immobilized DNA polymerase consisting essentially of a DNA polymerase, whose active site is masked with a DNA substrate, a linker, and a supporting material, wherein the DNA polymerase forms covalent bonding with at least one linker; the linker forms chemical bonding with the supporting material; the immobilization site of the DNA polymerase that will be connected to the linker is selected such that the DNA polymerase is oriented; and the average number of covalent bonding per immobilized DNA polymerase is controlled, such that the average activity of the immobilized DNA polymerase is preserved more than 10% relative to that of the solution phase DNA polymerase.
  • the immobilized DNA polymerase provided by the present invention has highly preserved activity, given the following two characteristics for covalently immobilizing the DNA polymerase on the supporting material through the linker.
  • an oriented immobilization is induced to form covalent bonding for immobilization at a site distant from the active site of the DNA polymerase so that the activity of the enzyme is preserved after covalent immobilization, order for the oriented immobilization to occur, the DNA polymerase is masked by a DNA substrate that selectively binds to the active site of the DNA polymerase. In this manner, formation of covalent bonding for immobilization at or near the active site of the DNA polymerase is prevented.
  • the DNA polymerase is immobilized in the same way as described above but without masking the enzyme, that is, the case where a random immobilization takes place, it is confirmed that the activity is almost lost after immobilization. This confirms that an oriented immobilization occurs when the enzyme is masked.
  • immobilization conditions are optimized to achieve high activity of the immobilized DNA polymerase.
  • concentration of an immobilization reaction group on the supporting material that forms a covalent immobilization bond with the DNA polymerase is controlled by changing the concentration of the linker that is chemically bound to the supporting material.
  • DNA polymerases per unit area of the supporting material should be high, (2) the number of covalent bonding for immobilization per immobilized DNA polymerase should be most preferably one, or else it should be maintained as minimal as possible, to the extent that the active site of the immobilized enzyme is not affected, if formation of multiple immobilization bonding is unavoidable under the given immobilization conditions.
  • the two conditions described above are contradictory each other.
  • the concentration of the immobilization reaction group should be increased.
  • the probability of forming a plurality of covalent bonding per immobilized DNA polymerase becomes high due to existence of too many immobilization reaction groups, and therefore the probability of losing the activity becomes high, h the opposite case where the concentration of the immobilization reaction group is too low, the number of covalent bonding for immobilization per immobilized DNA polymerase becomes low enough that the activity can be preserved.
  • the number of the immobilized DNA polymerase decreases, and therefore the overall activity can be reduced.
  • the activity of the immobilized DNA polymerase increases and then decreases as the concentration of the reaction group for immobilization on the supporting material increases. It is therefore demonstrated that construction of an immobilized DNA polymerase with an optimized high activity can be possibly provided, by compensating the two contradictory conditions.
  • PCR is performed with the immobilized DNA polymerase, and the result is compared with that obtained from the solution phase DNA polymerase wherein its amount corresponds to the maximum amount that can be immobilized on the supporting material.
  • the average activity of the immobilized DNA polymerase was determined relative to that of the solution phase DNA polymerase. Since the immobilized DNA polymerase of the present invention is likely to be a mixture of DNA polymerases that are immobilized in a different manner, the activity of the immobilized DNA polymerase is given as the average activity relative to that of the solution phase DNA polymerase.
  • the average activity of the immobilized DNA polymerase obtained in the present invention is more than 10% relative to the maximum activity of the solution phase DNA polymerase, and it is achieved to be as high as about 80% depending on the immobilization conditions. In addition, it is confirmed that the immobilized DNA polymerase can be recycled and reused after PCR.
  • the covalently immobilized DNA polymerase with highly preserved activity is realized in the present invention. At least in the case of the DNA polymerase, it is confirmed that there exists an immobilized state with the activity close to that of the solution phase DNA polymerase among various states of the immobilized DNA polymerase.
  • the Taq DNA polymerase used in the embodiments of the present invention is a protein with molecular weight of 94 kD that is composed of 832 amino acids.
  • the immobilization reaction group of the linker is carboxyl as in the embodiments of the present invention
  • there exist in the Taq DNA polymerase 146 amino acids having primary amines that can react with the carboxyl to form amide bonding.
  • the number of primary amines located outside the enzyme, which can form immobilization bonding is estimated to reach several tens.
  • the immobilization reaction is performed with a DNA polymerase with its active site masked by a DNA substrate that binds to the active site of the DNA polymerase. Due to this masking, the immobilization takes place in an oriented manner since immobilization reaction at or near the active site is prevented. Even though the immobilization occurs at a site relatively distant from the active site by the oriented immobilization in the manner described above, a plurality of covalent bonding for immobilization can be formed to damage the activity of the immobilized DNA polymerase since a plurality of immobilization reaction groups that can participate in the immobilization reaction exist on the surface of the DNA polymerase. Therefore, in the present invention, the average number of immobilization bonding per immobilized DNA polymerase can be controlled, by changing the concentration of the immobilization reaction group of the linker on the supporting material formed.
  • Example 4 it is observed that the average activity of the immobilized DNA polymerase relative to that of the solution phase DNA polymerase reaches maximum when the concentration of the carboxyl group used as the immobilization reaction group on the supporting material is 5%.
  • the average activity of the immobilized DNA polymerase increases rapidly as the concentration of the carboxyl group on the supporting material increases from 0% to 5%. This indicates that the number of immobilized DNA polymerases increases and the number of covalent bonding for immobilization per immobilized DNA polymerase is restricted to be one or as low as possible to preserve the activity.
  • the average activity of the immobilized DNA polymerase decreases rapidly as the concentration of the carboxyl group on the supporting material increases from 5% to 10%.
  • the structure of the most optimized immobilized DNA polymerase can be characterized in the following way.
  • the immobilized DNA polymerase of the present invention forms covalent bonding not at or near the active site, but at a site relatively distant from the active site, leading to an immobilized structure that is oriented.
  • the immobilized DNA polymerase provided in the present invention has a structure where the number of covalent bonding for immobilization per immobilized DNA polymerase is restricted to be one or as low as possible to avoid damaging the activity.
  • the immobilized DNA polymerase of the present invention can be constructed by selecting a DNA polymerase from the group consisting of thermostable DNA polymerases such as the Taq DNA polymerase and its derivatives, and DNA polymerases that are not thermostable, such as the E. coli DNA polymerase, the T7 DNA polymerase, and their derivatives.
  • thermostable DNA polymerases such as the Taq DNA polymerase and its derivatives
  • DNA polymerases that are not thermostable such as the E. coli DNA polymerase, the T7 DNA polymerase, and their derivatives.
  • the DNA polymerase consists of two protein domains that are structurally separated. The two domains are also separated functionally because only one of the domains has the activity for DNA polymerization. In order for DNA polymerization to occur, a partially double stranded DNA should bind to the domain having the activity for DNA polymerization.
  • the immobilized DNA polymerase of the present invention is constructed with an oriented immobilization that make it difficult to form covalent bonding for immobilization at the domain having the activity for DNA polymerization. This is because the active site of the DNA polymerase is masked with a partially double stranded DNA substrate.
  • the immobilized DNA polymerase of the present invention is constructed by forming covalent bonding for immobilization mainly at the other domain that does not have the activity for DNA polymerization. It will be obvious to those skilled in the art that instead of the Taq DNA polymerase, other DNA polymerases that have similar structures and functions to those of the Taq DNA polymerase can be used to construct immobilized DNA polymerases that have highly preserved activity, with the same structural characteristics.
  • the immobilized DNA polymerase of the present invention can be constructed using various linkers that are characterized in having reaction groups at both ends of the linker capable of binding to the DNA polymerase and the supporting material.
  • 12-mercaptododecanoic acid which has reaction groups at both ends of the 11 carbon chain, is used as a linker capable of binding to the DNA polymerase and the supporting material.
  • any material can be used if it has reaction groups at both ends capable of binding to the polymerase and the supporting material.
  • a linker that has a sufficient length so that interactions can be minimized between the immobilized DNA polymerase and the surface of the supporting material or the matrix formed on the surface. Therefore, in addition to the linker having a single alkane chain as used in the embodiments of the present invention, a linker comprising unsaturated carbon bonding, nitrogen, oxygen, sulfur, phosphorus, etc. or a linker having branched chain structure can be used.
  • the supporting material of the immobiHzed DNA polymerase of the present invention can be selected from the group consisting of metal, nonmetal, metalloid, their compounds, and their mixtures.
  • the supporting material can be constructed using a material having reaction groups or reaction sites capable of forming chemical bonding with a linker on its surface.
  • gold is used because immobilization conditions can be easily controlled.
  • any supporting material can be used if it has reaction groups or reaction sites capable of forming chemical bonding with the linker on its surface.
  • the immobilized DNA polymerase of the present invention can be constructed by forming covalent bonding between the DNA polymerase and the linker. It is particularly preferable to use amide bonding between primary amine and carboxyl groups, because they can react in mild conditions so as not to influence the enzyme structure.
  • proteins have various reaction groups such as amine, carboxyl, hydroxyl, imidazole, phenol, thiol, and indole. Therefore, the immobilization reaction can be selected by choosing a particular reaction group. However, it is preferable to select a reaction group that can react at a mild condition, compared to a reaction group whose reaction condition is so hard to affect the stability of the enzyme.
  • the immobilized DNA polymerase of the present invention is characterized in that a matrix molecule having a non-reactive terminal group bind to the supporting material in addition to the linker.
  • any matrix molecule can be used if it does not hamper the stability of the DNA polymerase.
  • the matrix molecule is used to control the concentration of the linker having the immobilization reaction group. More specifically, the concentration of the linker bound to the surface of the supporting material is controlled, by changing the relative concentration of the matrix molecule, hi addition to this function, it is possible to increase the activity preservation ratio or the stability of the DNA polymerase by selecting an appropriate matrix molecule.
  • the influence of the matrix molecule is shown in the embodiments of the present invention by comparing the two cases where 6-mercapto-l-hexanol and 1-heptanethiol are used as a matrix molecule.
  • Figure 1 is a diagram showing the construction of the immobilized DNA polymerase of the present invention
  • Figure 2 is a diagram showing the construction of the immobilized DNA polymerase with the active site masked by a DNA substrate of the present mvention
  • Figures 3 a and 3b are agarose gel electrophoresis photographs and a graph comparing the immobilized DNA polymerases immobilized according to the present invention and the prior method;
  • Figures 4a and 4b are an agarose gel electrophoresis photograph and a graph showing the activity change of the DNA polymerase depending on the characteristics of the matrix molecule;
  • Figure 5a and 5b are an agarose gel electrophoresis photograph and a graph showing that the immobilization site of the immobilized DNA polymerase of the present invention is oriented due to the masking of the active site;
  • Figure 6a and 6b are an agarose gel electrophoresis photograph and a graph showing the activity change of the immobilized DNA polymerase depending on the pH of the reaction between the DNA polymerase and the linker in the process of preparing the immobilized DNA polymerase of the present invention
  • Figure 7a and 7b are an agarose gel electrophoresis photograph and a graph showing the efficiency of DNA amplification depending on the number of PCR cycles when the immobilized DNA polymerase of the present invention is used in PCR.
  • Figures 8a and 8b are agarose gel electrophoresis photographs and a graph showing the results when the immobilized DNA polymerase is reused.
  • Figures 9a and 9b are an agarose gel electrophoresis photograph and a graph showing the stability of the immobilized DNA polymerase.
  • Example 1 Preparation of the immobilized DNA polymerase according to the present invention - PLM (Protected Immobilization Method)
  • the 65 base single stranded DNA (ss-DNA) and the KS primer shown below was mixed in an aqueous buffer solution at 1:1 molar ratio, and the resulting solution was incubated for 10 minutes at 94 "C and was then cooled down slowly below 35 ° C. During this process, the 65 base ss-DNA and the KS primer were annealed to form a partially double stranded DNA.
  • Taq DNA polymerase purchased from Perkin Elmer (U.S.A.) was then added to this solution and the resulting mixture was incubated in a dry bath at 72 ° C for 10 minutes. After this, the mixture was moved to a dry bath at 50 ° C and incubated for 20 minutes to prepare the PLM enzyme solution of the masked Taq DNA polymerase.
  • the Au substrate used was a glass plate of 3.0 mm X 5.0 mm size on which Au was vacuum-deposited to about 1000 A thickness, ha order to ensure the cleanness of the surface of the Au thin film, it was washed with Piranha solution for 10 ⁇ 15 minutes at 60 ⁇ 70 ° C right before using and was rinsed with deionized water and subsequently with absolute ethanol.
  • a monolayer of thiol molecules was formed on the Au surface by using the Au-S bond formation reaction, that is, by using the thiolate formation reaction between the linker having a thiol group and Au, to prepare a supporting material.
  • the mixed solution of two kinds of thiol molecules having an immobilization reaction group and a non-reactive group was used.
  • the mole fraction of thiol molecules having the immobilization reaction group was controlled by changing its mole fraction in the range 0 ⁇ 100%, in order to control the mole fraction of the immobilization reaction group on the supporting material.
  • 12-mercaptododecanoic acid with relatively longer alkyl chain was used.
  • 6-mercapto-1-hexanol or 1-heptanethiol was used as a thiol molecule having a non-reactive group.
  • the Au thin film was placed in 100 ⁇ l of a 2 mM mixed thiol solution in ethanol for 2 hours at room temperature to introduce the carboxyl immobilization reaction group, and it was washed with absolute ethanol.
  • the Au thin film where the carboxyl immobilization reaction group was introduced was placed in 120 ⁇ l of an ethanol solution containing 10 mM of l-ethyl-3-(3- dimetylaminopropyl)carbodiimide (EDC) and 5 mM of N-hydroxysuccinimide (NHS) for 2 hours at room temperature to activated the carboxyl group.
  • EDC l-ethyl-3-(3- dimetylaminopropyl)carbodiimide
  • NHS N-hydroxysuccinimide
  • the Au substrate was moved to the PPM enzyme solution.
  • the activated carboxyl (NHS-ester) on the monolayer reacted with the primary amine (-NH 2 ) of the protein to form amide bond (-CO-NH-).
  • the Taq DNA polymerase was immobilized on the supporting material.
  • the immobilization reaction was carried out at different conditions by varying concentration of the DNA polymerase, pH, reaction time, etc.
  • Figure 1 is a diagram showing the construction of the immobilized DNA polymerase prepared according to the present example.
  • the DNA polymerase 1 forms covalent bonding with the linker 2.
  • the primary amine of the polymerase forms amide bonding with the carboxyl of the linker.
  • the linker 2 is connected to the supporting material 3 via a Au-S chemical bonding. As in the present example, it can be constructed with or without introducing a matrix molecule 4 having a non-reactive group.
  • Figure 2 is a diagram showing the construction of the immobilized DNA polymerase prepared according to the present example.
  • the immobilized DNA polymerase shown in Figure 1 in which the DNA substrate used to mask the active site is removed, can be prepared by performing polymerization reaction with the immobilized DNA polymerase in Figure 2 in the presence of dNTP.
  • immobilization was performed by using a Taq DNA polymerase whose active site was not masked instead of the Taq DNA polymerase masked with the partially double stranded DNA substrate used in Example 1.
  • Other immobilization processes and reaction conditions were the same as in Example 1.
  • the activated Au substrate was placed in the RDVI enzyme solution to prepare an immobilized DNA polymerase.
  • Example 3 Measurement of the activity of the immobilized DNA polymerase
  • PCR was carried out, and the amount of the resulting amplified DNA was quantified.
  • PCR was carried out in the Model 480 PCR thermal cycler of Perkin Elmer.
  • the 65 bp ss-DNA shown in Example 1 was used as a template, and the KS primer and the SK primer (3'-CTAGGTGATCAAGATCT-5') were used as primers for PCR.
  • the volume of the PCR solution used was 50 ⁇ l.
  • Hot start step 94 ° C, 10 minutes
  • PCR For quantification of the DNA amplified by the PCR, 20 ⁇ l of the PCR solution was sampled and analyzed by agarose gel electrophoresis. The PCR products were then visualized by fluorescence from ethidium bromide staining and they are quantified with a densitometer.
  • Example 4 Activity of the immobilized Taq DNA polymerase as a function of the mole fraction of the carboxyl immobilization reaction group
  • the immobilization reaction was carried out in a phosphate buffer at pH 8.3 for 30 minutes at 50 ° C .
  • the immobilized Taq DNA polymerase was prepared according to Example 1 and 2 with 50 ⁇ l of the immobilization reaction solution containing 0.75 pmol of Taq DNA polymerase.
  • 0.75 pmol of the Taq DNA polymerase corresponds to the amount that can form three monolayers on the area of 3 mmX5 mm of the Au substrate used.
  • FIG. 3a shows the agarose gel fluorescence photograph of the PCR products.
  • the leftmost lane shows the ds-DNA molecular weight marker, and the rightmost lane shows the PCR product amplified with one monolayer amount of the solution phase Taq DNA polymerase.
  • Other lanes show the PCR products amplified by using the immobilized Taq DNA polymerase.
  • the numbers shown at the bottom are the mole fractions of 12- mercaptododecanoic acid relative to the total thiol molecules used for introducing the carboxyl group.
  • the activity determined from the agarose gel electrophoresis photographs in Figure 3a is depicted in Figure 3b.
  • the abscissa is the mole fraction of the thiol molecule having the carboxyl group relative to the total moles of the thiol molecules used.
  • the ordinate is the relative activity of the immobilized Taq DNA polymerase, as compared to the activity of one monolayer amount of the solution phase Taq DNA polymerase.
  • the PIM with the active site masking in which oriented immobilization thus took place shows higher activity than the RIM without the active site masking in which non-specific random immobilization took place. This demonstrates that the activity preservation of the PLM is much higher than that of the RLM because an oriented immobilization took place efficiently when the active site was masked .
  • the average activity of the immobilized DNA polymerase decreases rapidly as the concentration of the carboxyl group on the supporting material increases from 5% to 10%, although the number of immobilized DNA polymerases increases. This indicates that a considerable number of covalent bonding forms in most of the immobilized DNA polymerase, and thus the immobilized DNA polymerase is damaged, resulting in the reduction of the activity.
  • Example 5 Activity of the immobilized DNA polymerase depending on the matrix molecule
  • Figures 4a and 4b shows the results of the present example.
  • the numbers shown at the bottom of Figure 4a are the mole fraction of 12-mercaptododecanoic acid relative to the total number of thiol molecules used to introduce the carboxyl immobilization reaction group.
  • the activity is preserved above 10%, even though the matrix molecule is changed. Since the methyl non-reactive group introduced on the supporting material by 1-heptanethiol has physical and chemical properties that are different from the hydroxyl non-reactive group introduced by 6-mercapto-l-hexanol, the change of the activity observed as a function of the carboxyl mole fraction differs in the two cases.
  • Example 6 Activity of the immobilized DNA polymerase as a function of the masking ratio of the active site
  • the number of moles of the partially double stranded DNA used to mask the active site relative to that of the Taq DNA polymerase used was varied from 0 to 2, and the activity of the immobilized Tag DNA polymerase was measured .
  • the mole fraction of 12- mercaptododecanoic acid with respect to the total moles of the thiol molecules used for introducing the carboxyl immobilization reaction group on the Au surface was 5.0%.
  • the total amount of the Taq DNA polymerase used for the immobilization reaction was 0.75 pmol, which corresponded to three monolayers.
  • Other reaction conditions for immobilization and PCR were the same as in Example 4.
  • FIGS. 5a and 5b The results are shown in Figures 5a and 5b.
  • the activity of the immobilized enzyme is shown as the relative enzyme activity compared to that in solution.
  • the leftmost and rightmost lanes are the same as in Figure 3 a, and other lanes are the results of the PCR products amplified with the immobilized Taq DNA polymerases at different masking ratio.
  • the numbers given at the bottom are the ratio corresponding to the number of moles of the partially double stranded DNA used for masking relative to that of the Taq DNA polymerase used.
  • Example 7 Activity of the immobilized Taq DNA polymerase in the PLM as a function of the immobilization pH
  • the activity of the immobilized DNA polymerase was measured at different immobilization pH, while keeping the mole fraction of 12-mercaptododecanoic acid at 5.0% with respect to the total moles of the thiol molecules used for introducing the carboxyl immobilization reaction group on the Au surface.
  • Other reaction conditions for immobilization and PCR were the same as in Example 4.
  • Figures 6a and 6b show that the leftmost and rightmost lanes in Figure 6a are the same as in Figure 3 a, and other lanes are the results of the PCR products amplified with the immobilized Taq DNA polymerase at different pH shown at the bottom of each lane.
  • Figures 6a and 6b show that the activity of the immobilized Taq DNA polymerase changes depending on pH.
  • the activity of the immobilized Taq DNA polymerase is maximized at pH 8.3, where the binding activity of the Taq DNA polymerase is known to be maximum. This demonstrates again that the oriented immobilization induced by the active site masking is important for preserving the activity of the immobilized enzyme.
  • Example 8 Comparison of the activity of the solution phase and the immobilized Taq DNA polymerase prepared by PLM as a function of the number of PCR cycles
  • the activity of the immobilized Taq DNA polymerase was measured at different number of PCR cycles, while keeping the mole fraction of 12-mercaptododecanoic acid at 5.0% with respect to the total moles of the thiol molecules used for introducing the carboxyl immobilization reaction group on the Au surface.
  • Other reaction conditions for immobilization and PCR were the same as in Example 4.
  • DNA polymerase is nearly identical to that of the solution phase Taq DNA polymerase.
  • the mole fraction of 12-mercaptododecanoic acid with respect to the total moles of the thiol molecules used for introducing the carboxyl immobilization reaction group on the Au surface was kept at 5%, and all other reaction conditions for immobilization were the same as in Example 4.
  • the immobilized Taq DNA polymerase was taken out of the PCR tube after 35 cycles of PCR at the same conditions described at Example 3 and repeatedly used for new 35 cycles of PCR.
  • the product of each PCR was analyzed.
  • the results are shown in Figures 8a and 8b.
  • the numbers shown above the agarose gel electrophoresis photograph in Figure 8a are the numbers of the total PCR cycles.
  • the activity changes observed as a function of the number of PCR cycles show a similar pattern for both the solution phase and the immobilized Taq DNA polymerase prepared in the present example.
  • the immobilized DNA polymerase can be recycled and reused repeatedly.
  • the observed reduction of the activity as the PCR cycles increase is due to the fact that the activity reduction of the Taq DNA polymerase is caused by the damage induced by heat at above 90 ° C during the denaturation step in PCR. If the immobilized Taq DNA polymerase of the present invention is used in a polymerization reaction without a high temperature process, it can be reused for much more cycles.
  • Example 10 Stability test for storage of the immobilized DNA polymerase prepared by the PLM
  • the mole fraction of 12-mercaptododecanoic acid with respect to the total moles of the thiol molecules used for introducing the carboxyl immobilization reaction group on the Au surface was kept at 5%, and all other reaction conditions for immobilization were the same as in Example 4.
  • the immobilized Taq DNA polymerase was placed in a storage solution at 4 ° C .
  • the storage solution was a 20 mM tris buffer at pH 9.0 containing 100 mM potassium chloride, 0.1 mM EDTA, 1.0 mM DTT (dithiothreitol), 0.5% Tween 20, and 50% glycerol. After each storage period, the activity of the immobilized Taq DNA polymerase was measured by performing 35 cycles of PCR as described in Example 3.
  • Figures 9a and 9b show the activity change of the immobilized Taq DNA polymerase depending on the storage period.
  • the numbers shown at the upper side of Figure 9a are storage periods. The results show that about 70% of the activity relative to the initial activity can be maintained even after the storage period of 2 month. This confirms that the immobilized DNA polymerase of the present invention is stable enough that it can be stored for a long period and reused.
  • the immobilized DNA polymerase of the present invention can preserve its activity from more than 10% to preferably 80%, under the conditions that the enzyme is immobilized with an oriented manner so as to form covalent bonding at a site other than the active site and also that the number of covalent bonding for immobilization is restricted to be one or an appropriate number to provide a desired structure for the immobilized enzyme.
  • the present invention provides the immobilized DNA polymerase that is constructed to have highly preserved activity, separation and recycling of the DNA polymerase can be performed easily after the enzyme reaction.
  • the immobilized DNA polymerase provided by the present invention thus make it possible to reuse the DNA polymerase and to simplify the purification process for the enzyme reaction sample. Therefore, it is possible to increase efficiencies of enzyme reaction processes and apparatuses in which the immobilized DNA polymerase that can simplify the separation process for the enzyme is used.

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Abstract

ADN polymérase immobilisée par liaison covalente, qui conserve une activité élevée et dont le site actif est masqué par un substrat d'ADN. L'ADN polymérase immobilisée selon la présente invention est constituée essentiellement d'une ADN polymérase, d'un adaptateur, et d'une matière de support. Ladite ADN polymérase forme une liaison covalente avec au moins un adaptateur, l'adaptateur forme une liaison chimique avec la matière de support, le site d'immobilisation de l'ADN polymérase qui sera connecté à l'adaptateur est choisi de sorte que l'ADN polymérase soit orientée, et le nombre moyen de liaisons covalentes par ADN polymérase immobilisée est régulé de sorte que l'activité moyenne de l'ADN polymérase immobilisée soit préservée à plus de 10 % par rapport à celle de l'ADN polymérase en phase de solution.
PCT/KR2001/001650 2000-10-04 2001-09-29 Adn polymerase immobilisee WO2002029027A1 (fr)

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AU2001294299A AU2001294299A1 (en) 2000-10-04 2001-09-29 Immobilized dna polymerase
US10/406,154 US7238505B2 (en) 2000-10-04 2003-04-02 Immobilized DNA polymerase
US11/809,188 US8067174B1 (en) 2000-10-04 2007-05-30 Polymerase chain reaction (PCR) method for amplifying a DNA template

Applications Claiming Priority (4)

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KRPCT/KR00/01104 2000-10-04
PCT/KR2000/001104 WO2002074993A1 (fr) 2000-10-04 2000-10-04 Procede permettant l'immobilisation de molecules presentant une activite physiologique
PCT/KR2001/001239 WO2003008570A1 (fr) 2001-07-20 2001-07-20 Procede pour l'immobilisation de molecules ayant une activite physiologique
KRPCT/KR01/01239 2001-07-20

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US7238505B2 (en) 2000-10-04 2007-07-03 Ahram Biosystems Inc. Immobilized DNA polymerase
KR100740869B1 (ko) 2002-09-13 2007-07-19 아람 바이오시스템 주식회사 고정화된 디엔에이 중합효소를 사용한 염기서열 증폭 방법및 장치
US7452669B2 (en) * 2003-10-30 2008-11-18 Samsung Electronics Co., Ltd. Micro PCR device, method of amplifying nucleic acid and method of measuring concentration of PCR product using the same
US7998672B2 (en) 2006-05-30 2011-08-16 The University Of Utah Research Foundation Simultaneous amplification and detection of ribonucleic acid be an optical method using surface plasmon resonance
AU2006331512B2 (en) * 2005-12-22 2012-02-23 Pacific Biosciences Of California, Inc. Active surface coupled polymerases
US8921086B2 (en) 2005-12-22 2014-12-30 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
CN108130317A (zh) * 2016-12-01 2018-06-08 苏州百源基因技术有限公司 一种dna聚合酶的固定化方法及其应用

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CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 98:68470G, SAGI J. ET AL. XP002907168 *
IBORRA F.J. ET AL.: "The topology of transcription by immobilized polymerases", EXP. CELL RES., vol. 229, no. 2, 1996, pages 167 - 173 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8067174B1 (en) 2000-10-04 2011-11-29 Ahram Biosystems Inc. Polymerase chain reaction (PCR) method for amplifying a DNA template
US7238505B2 (en) 2000-10-04 2007-07-03 Ahram Biosystems Inc. Immobilized DNA polymerase
KR100740869B1 (ko) 2002-09-13 2007-07-19 아람 바이오시스템 주식회사 고정화된 디엔에이 중합효소를 사용한 염기서열 증폭 방법및 장치
US7452669B2 (en) * 2003-10-30 2008-11-18 Samsung Electronics Co., Ltd. Micro PCR device, method of amplifying nucleic acid and method of measuring concentration of PCR product using the same
US9951321B2 (en) 2005-12-22 2018-04-24 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
AU2006331512B2 (en) * 2005-12-22 2012-02-23 Pacific Biosciences Of California, Inc. Active surface coupled polymerases
US8921086B2 (en) 2005-12-22 2014-12-30 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
US8936926B2 (en) 2005-12-22 2015-01-20 Pacific Biosciences Of California Active surface coupled polymerases
US9556479B2 (en) 2005-12-22 2017-01-31 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
US10717968B2 (en) 2005-12-22 2020-07-21 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
US11299720B2 (en) 2005-12-22 2022-04-12 Pacific Biosciences Of California, Inc. Polymerases for nucleotide analogue incorporation
US7998672B2 (en) 2006-05-30 2011-08-16 The University Of Utah Research Foundation Simultaneous amplification and detection of ribonucleic acid be an optical method using surface plasmon resonance
CN108130317A (zh) * 2016-12-01 2018-06-08 苏州百源基因技术有限公司 一种dna聚合酶的固定化方法及其应用
CN108130317B (zh) * 2016-12-01 2020-05-26 苏州百源基因技术有限公司 一种dna聚合酶的固定化方法及其应用

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