WO2003027238A2 - Stabilization of t4 endonuclease v - Google Patents
Stabilization of t4 endonuclease v Download PDFInfo
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- WO2003027238A2 WO2003027238A2 PCT/US2002/029885 US0229885W WO03027238A2 WO 2003027238 A2 WO2003027238 A2 WO 2003027238A2 US 0229885 W US0229885 W US 0229885W WO 03027238 A2 WO03027238 A2 WO 03027238A2
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- endonuclease
- activity
- composition
- protease activity
- peptide
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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
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
Definitions
- the present invention relates to preparations of the enzyme T4 endonuclease V that are stable during storage at temperatures above refrigeration.
- cold is “any temperature not exceeding 8°C (46°F)” and a “refrigerator is a cold place in which the temperature is maintained thermostatically between 2° and 8°C (36° and 46°F).” Accordingly, as used herein, “refrigeration” refers to temperatures up to 8°C (46°F).
- T4 endonuclease V (referred to herein as
- T4 endo V is useful in medicine, health maintenance, and for other purposes. Its use, however, has been limited by its instability when stored at temperatures above refrigeration.
- the present invention describes methods for providing T4 endo V with stability at temperatures above refrigeration.
- the invention also provides (1) assay methods for use in achieving this stability and (2) T4 endo V compositions which exhibit such stability.
- U.S. Patent #5,077,211 and U.S. Patent # 5,272,079, both entitled “Purification and Administration of DNA Repair Enzymes”, relate to methods for purifying DNA repair enzymes and encapsulating them in liposomes for topical delivery.
- U.S. Patent #5,296,231 "Purification and Administration of DNA Repair Enzymes” relates to methods for purifying DNA repair enzymes and encapsulating them in liposomes sensitive to changes in pH for purposes of topical delivery.
- U.S. Patent #5,190,762 “Method of Administering Proteins to Living Skin Cells” relates to the application of biologically active proteins, including enzymes, in liposomes into cells of the skin.
- Endonuclease relates to the use of DNA repair liposomes encapsulating T4 endo V to protect the immune system.
- U.S. Patent #5,352,458 "Tanning Method Using DNA Repair Liposomes" relates to the use of DNA repair liposomes to increase the tanning response.
- T4 endonuclease V Mutation Research. (1987) volume 183, pp. 117-121. A method for purification of T4 endonuclease V is described. All procedures are recommended to be performed at 0-4°C unless otherwise noted. No recommendation on storage is noted.
- T4 endo V contains both a dimer specific glycosylase activity and an AP endonuclease activity on the same polypeptide.
- the AP DNA endonuclease activity was destroyed at 42°C very rapidly, within 5 minutes, while the glycosylase activity was lost at a rate of 4% per minute at this temperature.
- T4 endonuclease V Further purification of T4 endonuclease by several procedures has been reported; however, in our experience, extensively purified enzyme is labile to storage.” Yarosh, D., A. O'Connor, L. Alas, C. Potten, P. Wolf. Photoprotection by topical DNA repair enzymes: Molecular correlates of clinical studies. Photochemistrv and Photobiology. (1999) 69:136-140. Inactivation of the T4 endo V was measured in some lots at 10°C, 25°C and 37°C. The enzyme is shown to recover activity under certain conditions after SDS denaturation as well as heat denaturation. The hypothesis is presented that the lability of T4 endonuclease V is due to the ease of protein unfolding at the hinge regions between the helices.
- T4 endonuclease V Biochimica et Biophvsica Acta (1976) volume 442, pp. 197-207.
- a method for purification of T4 endonuclease V is described. All procedures are recommended to be carried out at 0-4°C. The preparation used in most of the experiments was stored at 0°C (page 202).
- the enzyme is noted to be labile: "Although endonuclease V is relatively stable when stored at 0°C it is extremely heat labile and loses 80% of its activity within 8 minutes of preincubation at 40°C.” (page 203-204)
- T4 endo V As mentioned above, a significant impediment to the widespread use of T4 endo V is the current limitation on its storage at temperatures greater than refrigeration. Many methods have been used to purify T4 endo V, including the methods described in the references referred to above. Some of these methods have included the use of metal chelators such as EDTA and/or ion-exchange chromatography. However, commercial application of these methods, e.g., application of the methods to fermentations and purifications of 200 liters or more, yield products that are not stable for periods of a week or longer unless stored at refrigeration or frozen. Stability studies presented in the literature demonstrate a short half-life for T4 endo V at temperatures greater than refrigeration. Commercial suppliers of T4 endo V, such as EPICENTRE of Madison, Wisconsin always ship their enzymes under refrigerated or frozen conditions, despite the additional costs, and recommend storage under frozen conditions.
- EPICENTRE of Madison, Wisconsin always ship their enzymes under refrigerated or frozen conditions, despite the additional costs, and
- the present invention provides a composition of T4 endo V that can be stored at greater than refrigerated temperature (e.g., at room temperature). This is accomplished by removal of cryptic (trace) proteases and/or the inhibition of such protease activity.
- the present invention provides a method for storing T4 endo V at greater than refrigerated temperature by including in the composition protease inhibitors, deflectors of protease activity, or decoys for protease activity.
- T4 endo V that can be stored at greater than refrigerated temperatures is produced by removing proteases during enzyme manufacturing or by including in the manufacturing process protease-specific proteases, suicide inhibitors of proteases and/or chemical inactivators of proteases in amounts sufficient to eliminate the trace protease activity that remains after standard purification.
- the present invention further provides a method for detecting trace protease activity not otherwise detected by extending the time in which an indicator protease substrate (reporter peptide) is in contact with a test sample.
- the invention provides a method for detecting protease contaminants in a T4 endonuclease V composition (e.g., an intermediate composition in an overall manufacturing process or a final commercial composition) comprising:
- test solution comprising an aqueous buffer (e.g., a Tris-HCl, pH 8 buffer), a sample of the T4 endonuclease V composition, and a reporter peptide; (ii) incubating (under standard laboratory conditions) the test solution at 25°C for a period of at least 7 days; and
- aqueous buffer e.g., a Tris-HCl, pH 8 buffer
- the concentration of the T4 endonuclease V in the test solution is 0.095 ⁇ 0.05 mg/ml
- the reporter peptide is a labeled reporter peptide whose amino acid sequence is Pro Leu Ser Arg Thr Leu Ser Val Ala Ala Lys (i.e., the Cl reporter peptide of the commercial PROMEGA PEPTAGTM kit (PROMEGA Corporation (Madison, Wisconsin)); see SEQ ID NO: 1), and/or (3) step (iii) is performed by subjecting at least a portion of the test solution to a peptide separation procedure (e.g., agarose gel electrophoresis) and determining the percentage of degradation of the reporter peptide by densitometry (e.g., a computer analysis of a digitized fluorescent image of the gel for a reporter peptide having a fluorescent label).
- a peptide separation procedure e.g., agarose gel electrophoresis
- densitometry e.g., a computer analysis of a digitized fluorescent image of the gel for a reporter peptide having a fluorescent label.
- positive and negative controls are preferably also tested.
- the positive control will contain a protease known to degrade the labeled reporter peptide used in the test, while the negative control can be, for example, the buffer and the labeled reporter peptide with no sample of the composition.
- the concentration of the reporter peptide in the test solution of step (i) is selected so that the protease activity of the positive control can be reliably detected.
- suitable reporter peptide concentrations in the test solution include 0.03 mg/ml, 0.04 mg/ml, 0.06 mg/ml, and 0.095 mg/ml, each of which will reliably detect protease activity in a positive control, such as, the alkaline protease positive control recommended for use with the PROMEGA PEPTAGTM kit. See Promega Corporation's Technical Bulletin No.
- reporter peptide concentrations can, of course, be used in the practice of the invention, e.g., any concentration in, for example, the 0.03 to 0.095 mg/ml range or the 0.04 to 0.06 mg/ml range, provided the concentration is sufficiently high to reliably detect the presence of proteases in a positive control under the particular assay conditions employed.
- the reporter peptide/positive control/negative control combination results in 100% degradation of the reporter peptide by the positive control and 0% degradation by the negative control under the assay conditions employed.
- the concentration of the reporter peptide in the test solution should be greater than 0.03 mg/ml.
- a method for preparing a T4 endonuclease V composition e.g., an intermediate composition in an overall manufacturing process or a final commercial composition
- step (B) treating the aqueous solution so as to reduce the protease activity of the composition (e.g., through the use of a quarternary ammonium moiety attached to a solid support and/or with a chelating agent); wherein as a result of step (B), the composition has a protease activity of less than 10 percent, where the protease activity is determined by the first method aspect of the invention employing, as discussed above, (1) the preferred T4 endonuclease V concentration, (2) the preferred reporter peptide, and (3) the preferred degradation determination technique, the percentage of degradation so determined being the protease activity.
- the invention provides a composition comprising T4 endonuclease V wherein the composition has a protease activity of less than 10 percent, where the protease activity is determined by the first method aspect of the invention employing, as discussed above, (1) the preferred T4 endonuclease V concentration, (2) the preferred reporter peptide, and (3) the preferred degradation determination technique, the percentage of degradation so determined being the protease activity.
- the invention provides a composition comprising T4 endonuclease V (e.g., a composition of T4 endonuclease V and a suitable carrier such as an aqueous carrier containing pharmaceutical excipients) wherein the composition retains at least 10 percent of its initial activity after storage at 25°C for a period of six months.
- T4 endonuclease V e.g., a composition of T4 endonuclease V and a suitable carrier such as an aqueous carrier containing pharmaceutical excipients
- Figure 1 shows the relation between initial activity and rate of decay of T4 endonuclease V. This graph plots the slope of the stability curve against the initial activity for three lots of T4 endonuclease V.
- the data was derived from stability studies, in which the slope of the stability curve is the slope of a linear regression line fitted to a plot of T4 endonuclease V activity versus time, and the initial activity is taken as the intercept obtained when the regression equation is solved for the date of manufacturing of the enzyme.
- the fit of the regression line to the data of this figure has an R 2 value of 0.9093.
- Figure 3 shows the relationship of protease activity to stability of T4 endonuclease V.
- the two lots differed in that Lot I was prepared from the central portion of the single- stranded DNA elution peak, and Lot H was prepared from the flanking portions of this central portion of the elution peak.
- the protease activity was determined using the Cl peptide at a concentration of 0.06 mg/ml in the test solution.
- Figure 4 shows partial inhibition of protease activity by EDTA.
- Two lots of T4 endo V, Lot C and Lot I were tested for protease activity without and with increasing concentrations of EDTA. Neither preparation showed protease activity when tested with the Al peptide at a concentration of 0.06 mg/ml in the test solution.
- Assays using the Cl peptide at the same concentration revealed that Lot C had a lower level of protease activity than Lot I, and that this was completely inhibited by the addition of EDTA.
- Lot I had greater protease activity that was also partially inhibited by EDTA.
- Figure 5 shows partial removal of protease activity by fast-flow Q-SEPHAROSE (FFQS).
- Crude T4 endonuclease V lysate was passed over a column of Q-SEPHAROSE, and the protease activity was compared using both the Al and Cl peptides incubated for 24 hrs with the concentration of reporter peptide in the test solution in each case being 0.04 mg/ml.
- the preparation Prior to application to the column, the preparation had protease activity against either peptide. After flow-through, the levels of protease activity were greatly reduced.
- Figure 6 shows removal of protease activity in T4 endonuclease V lots containing EDTA and passed through FFQS.
- Figure 7 shows stability at 25°C with protease removal.
- FIG 8 shows that T4 endo V (Lot M) prepared with inclusion of 3.4 mM EDTA and passage over a column of Q-SEPHAROSE contained substantially less protease than batches made without EDTA or Q-SEPHAROSE (Lot H & I), or a commercially available batch of T4 endo V from EPICENTRE (Madison, WI).
- the protease assay was done by incubating test materials with Cl peptide at 25°C and 37°C for 2 days. The concentration of Cl peptide in the test solution was 0.03 mg/ml.
- the present invention provides T4 endo V compositions that can be stored at temperatures above refrigeration by the elimination of cryptic proteases from the compositions.
- protease activity was assayed in lots of T4 endo V using a standard protease activity assay, namely, the PEPTAGTM assay of PROMEGA Corporation (Madison, Wisconsin).
- a standard protease activity assay namely, the PEPTAGTM assay of PROMEGA Corporation (Madison, Wisconsin).
- peptides with fluorescent end-labels are incubated with a test sample, and then the peptides are subjected to electrophoresis. If protease activity cleaves the peptides, the migration of the end-labeled peptide is altered because it is shorter and/or changed in charge.
- Two test peptides of different lengths and amino acid sequences are used to detect a broader range of proteases.
- the level of protease activity was then analyzed in two of the T4 endo V preparations, and compared to the stability of those preparations.
- the slope of the enzyme activity versus time curve was found to correlate with protease activity. In particular, the lots with the higher protease activity had the least stability, and the lots with the least protease activity had the greatest stability.
- protease activity is enzymes that degrade protein. They can be non-specific, and attack almost all proteins, or specific for certain proteins or certain peptide linkages. A species of proteases is often found in nature within a mixture of several proteases. In some cases enzymes are reported to be sensitive to cold or freezing, but this can be due to protease contaminants in the preparation that have time to act during the thawing process when such a process is performed at an elevated temperature. Proteases are often stimulated by using divalent ions as co-factors, and therefore the chelating activity of EDTA can reduce protease activity.
- EDTA is generally used as an efficient chelator in biochemistry at concentrations above 0.1% and below 0.5 wt.%. EDTA may be kept in the final product or removed if desired. There are several compounds similar in structure and activity to EDTA, such as EGTA and ergothioneine. In accordance with the invention, the concentration of chelating agent must be high enough to significantly reduce cryptic protease activity, e.g., reduce such activity by at least 50%, preferably by 80%, and most preferably by 90%.
- An additional method for reducing protease activity is to use molecules that disrupt or block the active site or mechanical functions of the protease. They may be specific for a protease, or broad-spectrum inhibitors.
- Molecules such as these include phenylmethylsulfonyl fluoride and leupeptin.
- the preferred protease inhibitor is non-toxic, active at low concentrations, inexpensive, and does not interfere with the activity of the T4 endo V enzyme. Such inhibitors do not remove, but rather merely inhibit, proteases, and therefore, when used, they are included in the final enzyme preparation.
- chelators can be removed from the final preparation because they take with them protease co-factors (e.g., divalent ions) and thus in this way reduce protease activity.
- concentration of the inhibitor must be high enough to significantly reduce cryptic protease activity, e.g., reduce such activity by at least 50%, preferably by 80%, and most preferably by 90%.
- a preferred approach for reducing protease activity in T4 endo V compositions is to remove proteases through binding to quaternary ammonium groups ("Q" groups). Such removal can be accomplished by means of Q-based chromatography media, such as Q-SEPHAROSE and fast flow Q membranes.
- Q-based chromatography media such as Q-SEPHAROSE and fast flow Q membranes.
- Q-SEPHAROSE quaternary ammonium groups
- Q-based chromatography media such as Q-SEPHAROSE and fast flow Q membranes.
- Q-based chromatography media such as Q-SEPHAROSE and fast flow Q membranes.
- Q and Q- are used herein to refer to all affinity binding media with anion exchange characteristics similar to quaternary ammonium moieties. These media can be used in column chromatography, batch stirring, or solid support forms.
- Q-binding is less than that of T4 endo V, then protease activity will be found in the flow-through fractions, and the T4 endo V without proteases can be eluted from the Q-binding matrix, using salt or pH or other chaotropic agents.
- the most desirable process is one that uses inexpensive chromatography media that is easily cleaned, and a process where T4 endo V flows through and proteases are retained.
- Q-binding preferably significantly reduces cryptic protease activity of the T4 endo V composition, e.g., reduces such activity by at least 50%, preferably by 80%, and most preferably by 90%.
- Several other methods are available for reducing or eliminating protease activity in T4 endo V compositions.
- Molecular chaperones are protein molecules that bind to a wide variety of enzymes and protect them from denaturation and others hazards within the cell. Chaperones or other physical protectors from proteases may be used to increase the stability of T4 endo V. The most desirable chaperones are those that are inexpensive, non-toxic, and do not inhibit the activity of T4 endo V. Decoy substrate proteins, and suicide protease substrates may be used to absorb, sequester or destroy proteases. These have the advantage of destroying the protease activity so that the proteases can be subsequently removed. The preferred decoys and suicide substrates are those that are efficient, inexpensive, non- toxic and easily removed. Again, these methods should achieve a significant reduction in protease activity, e.g., a reduction in such activity by at least 50%, preferably by 80%, and most preferably by 90%.
- the T4 endo V used in the practice of the invention may exist in an extract, or in a semi-pure or purified state prepared from feedstock that contains proteases.
- the enzyme may be fermented from either prokaryotic or eukaryotic cells, including plants.
- the final T4 endo V composition can include various components conventionally used in the pharmaceutical and cosmetic fields, such as excipients, anti-microbials, bulking agents, coloring agents, fragrances, and the like.
- the T4 endo V compositions of the invention will be used in topical preparations applied to humans or animals.
- the T4 endo V composition will comprise liposomes in which the enzyme is encapsulated.
- Example 1 This example demonstrates that the relationship between T4 endo V enzyme stability and initial activity indicates the presence of protease activity, even if protease activity cannot be detected by standard assays. The relationship can be seen in a plot of three or more lots of the enzyme preparation, when the slope of the loss of enzyme activity is plotted against the initial enzyme activity.
- T4 endonuclease activity was determined by preparing a substrate of plasmid pMJRl560 at 200 ⁇ g/ml irradiated with 40 J/m 2 of UV-C from a germicidal lamp, and mixing it with 200 ⁇ g/ml of unirradiated plasmid pSV2neo. Ten ⁇ l of this substrate was then mixed with 1 ng of each of three lots of T4 endonuclease V protein in a 50 mM Tris-HCl buffer, pH 8 and 10 mM EDTA. The lots had been purified in accordance with the procedures of U.S. Patent # 5,077,211. (The procedures of this patent were also used for preparation of all of the other lots referred to herein except where indicated.) After 60 min incubation at 37°C, the DNA was analyzed on a 0.8% neutral agarose gel.
- the substrate migrates as two supercoiled bands, with the irradiated and smaller pMJR1560 plasmid migrating faster than the larger pSV2neo plasmid.
- T4 endonuclease V acts on the UV-irradiated supercoiled plasmid pMJR1560
- the resulting open circular plasmid form migrates more slowly than the pSV2neo plasmid.
- the percent of the total pMJR1560 plasmid that migrates in the supercoiled position is an inverse measure of T4 endonuclease V activity.
- One "hit" is the amount of enzyme activity that reduces the amount of supercoiled plasmid to 37%.
- a unit of activity is equal to the number of hits for 100 ⁇ g of substrate mixture. Specific activity is the number of units per ⁇ g of enzyme.
- T4 endonuclease V were assayed during storage at 4°C.
- the data were plotted as net pMJR hits (by subtracting the hits found in the unirradiated control pSV2neo plasmid) versus date. From a plot of weekly measurements in duplicate over five months, a regression line was fitted to the data. The slope was taken as the slope of the stability curve, and each regression equation was solved for the activity at the initial date of manufacturing. The slope of the stability curve was plotted against the initial activity. The result is shown in Figure 1. The resulting plot shows a linear relationship between slope and initial activity with a goodness of fit of 0.9093.
- Example 2 Standard assays for protease activity, such as the PROMEGA
- PEPTAGTM kit direct the user to incubate 1 ⁇ l of each of the Al and Cl substrates with 5 ⁇ l of the sample in a 10 ⁇ l total reaction buffered with 50 mM Tris-HCl, pH 8.0, for 30 minutes at room temperature, and then to analyze the products in a 1% agarose gel.
- a positive control 2.5 ⁇ l of a 20ng/ ⁇ l solution of alkaline protease is used, i.e., 50 ng of alkaline protease. Under these conditions, no protease activity was detected in 9 lots of T4 endonuclease V assayed at 30 minutes or even up to 18 hours ( Figure 2).
- Example 3 Two lots of T4 endo V were prepared from the same gel filtration process intermediate but were split into two parts based on elution from the column and were designated Lot I which was the central portion of the elution peak and Lot H which was a combination of the flanking fractions of the elution peak.
- Example 4 This example illustrates the use of ethylenediaminetetracetic acid (EDTA) to inhibit protease activity in preparations of T4 endo V.
- EDTA ethylenediaminetetracetic acid
- Two lots were selected with high and low protease activity ( Figure 4).
- EDTA ethylenediaminetetracetic acid
- Lot C degraded only 28% of the Cl peptide at 25°C
- Lot I degraded 100% of the peptide.
- Addition of 0.1% to 1.5% (w/v) EDTA significantly reduced the protease activity in Lot C, while 0.1% to 1.5% EDTA only reduced the cleavage of the Cl peptide by about 50% in Lot I.
- the Al peptide did not reveal this finding ( Figure 4) since it did not consistently exhibit substantial degradation by the protease or proteases of the T4 endo V solution.
- Example 5 This example illustrates the use of anion exchange chromatography to remove protease activity during enzyme purification.
- Fast-flow Q-SEPHAROSE BIORAD, Richmond, CA
- phosphate buffered saline, pH 7.8 Two hundred ml of cell lysate containing T4 endonuclease V was divided into two parts and each part was passed over the FFQS column and the flow through was collected. The column was cleaned with high salt and 1 M NaOH between passages.
- FIG. 5 shows that the protease activity was substantially reduced by this treatment, in a protease assay of 24 h.
- the lysates contained protease activity both before filtration (FFQS-Pre-0.2 ⁇ m filtration) and after filtration just prior to loading the column (FFQS-Load).
- the flow-through fractions which contained the enzyme activity, showed greatly reduced protease activity, i.e., the proteases remained bound to FFQS. This was observed with both the Al and the Cl peptide. Both these reporter peptides were able to detect protease activity because of the impure state of the lysate and the high concentrations of protein. For the same reasons, a 24 hour incubation revealed the protease activity.
- Example 6 shows that the protease activity was substantially reduced by this treatment, in a protease assay of 24 h.
- the lysates contained protease activity both before filtration (FFQS-Pre-0.2 ⁇
- T4 endonuclease V was prepared using the method described in U.S. Patent #5,077,211 and also included two additional treatment steps, namely, a Q-SEPHAROSE treatment step after cell lysis and addition of 3.4 mM EDTA during subsequent purification steps.
- T4 endo V (Lot K) was prepared using the techniques of Example 6. The final product was then tested for stability at room temperature (25°C) versus stability at refrigeration (4°C). No difference in the specific activity of the protein, determined by statistical analysis, could be found after storage for 7 days (see Figure 7).
- T4 endo V (Lot L) was prepared using the same method, and the final product was compared to T4 endo V prepared without removal/inhibition of proteases (Lot G of Figure 2).
- the enzymes were held at 25°C and assayed at the times indicated in Table 1. The activity at each time point was compared to the activity at the start. No T4 endo V activity was detected in the preparation with proteases after 1 week of incubation, i.e., Lot G of Figure 2. In the protease-reduced preparation, T4 endo V activity was reduced but measurable after the first week, and no further activity was lost over the course of the next 3 weeks.
- Example 6 including passage of extracts over a column of Q-SEPHAROSE and the inclusion of 3.4 mM EDTA throughout the purification process.
- T4 endo V Lots H and I were prepared without Q-SEPHAROSE or EDTA.
- a commercially produced sample of T4 endo V was obtained from
- EPICENTRE (Madison, WI). These samples were then tested for protease activity during incubation with Cl peptide substrate for 2 days at 25°C and 37°C (see Figure 8). Because of the low concentration of T4 endo V supplied by EPICENTRE, the enzyme concentration in the assay for the EPICENTRE preparation was about one-tenth that specified above for the preferred protease assays of the present invention. When tested at 37°C, T4 endo V Lot M made with EDTA and Q-SEPHAROSE contained no detectable protease.
- T4 endo V Lots H and I made without EDTA and Q-SEPHAROSE contained low but clearly detectable protease activity
Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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EP02778299A EP1430143A4 (en) | 2001-09-24 | 2002-09-20 | Stabilization of t4 endonuclease v |
AU2002339973A AU2002339973B2 (en) | 2001-09-24 | 2002-09-20 | Stabilization of T4 endonuclease V |
CA002461019A CA2461019A1 (en) | 2001-09-24 | 2002-09-20 | Stabilization of t4 endonuclease v |
US10/490,397 US7094560B2 (en) | 2001-09-24 | 2002-09-20 | Stabilization of T4 endonuclease V |
JP2003530810A JP4283111B2 (en) | 2001-09-24 | 2002-09-20 | Stabilization of T4 endonuclease |
US11/507,213 US20060281166A1 (en) | 2001-09-24 | 2006-08-21 | Stabilization of T4 endonuclease V |
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US32444801P | 2001-09-24 | 2001-09-24 | |
US60/324,448 | 2001-09-24 |
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US11/507,213 Continuation US20060281166A1 (en) | 2001-09-24 | 2006-08-21 | Stabilization of T4 endonuclease V |
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Citations (1)
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US5272079A (en) * | 1988-07-06 | 1993-12-21 | Applied Genetics, Inc. | Purification and administration of DNA repair enzymes |
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US5190762A (en) * | 1988-07-06 | 1993-03-02 | Applied Genetics, Inc. | Method of administering proteins to living skin cells |
US5077211A (en) * | 1988-07-06 | 1991-12-31 | Applied Genetics, Inc. | Purification and administration of dna repair enzymes |
US5141852A (en) * | 1988-12-12 | 1992-08-25 | The United States Of America As Represented By The Department Of Health And Human Services | Assay of protein kinases with peptide substrates |
US5616311A (en) * | 1991-01-15 | 1997-04-01 | Hemosphere, Inc. | Non-crosslinked protein particles for therapeutic and diagnostic use |
EP0646242A4 (en) * | 1991-11-12 | 1997-12-03 | Promega Corp | Non-radioactive enzyme assay. |
US5302389A (en) * | 1992-08-17 | 1994-04-12 | Board Of Regents, The University Of Texas System | Method for treating UV-induced suppression of contact hypersensitivity by administration of T4 endonuclease |
US5352458A (en) * | 1992-12-21 | 1994-10-04 | Applied Genetics Inc. | Tanning method using DNA repair liposomes |
-
2002
- 2002-09-20 JP JP2003530810A patent/JP4283111B2/en not_active Expired - Fee Related
- 2002-09-20 WO PCT/US2002/029885 patent/WO2003027238A2/en active Application Filing
- 2002-09-20 AU AU2002339973A patent/AU2002339973B2/en not_active Ceased
- 2002-09-20 US US10/490,397 patent/US7094560B2/en not_active Expired - Fee Related
- 2002-09-20 CA CA002461019A patent/CA2461019A1/en not_active Abandoned
- 2002-09-20 EP EP02778299A patent/EP1430143A4/en not_active Withdrawn
-
2006
- 2006-08-21 US US11/507,213 patent/US20060281166A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5272079A (en) * | 1988-07-06 | 1993-12-21 | Applied Genetics, Inc. | Purification and administration of DNA repair enzymes |
Non-Patent Citations (3)
Title |
---|
DODSON M.L. ET AL.: 'Site-directed deletion mutagenesis within the T4 endonuclease V gene: dispensable sequences within putative loop regions' MUTA. RES., DNA REPAIR vol. 255, 1991, pages 19 - 29, XP002965458 * |
See also references of EP1430143A2 * |
YAROSH D.B. ET AL.: 'Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies' PHOTOCHEM. PHOTOBIOL. vol. 69, no. 2, 1999, pages 136 - 140, XP002965457 * |
Also Published As
Publication number | Publication date |
---|---|
EP1430143A4 (en) | 2005-07-13 |
JP2005503806A (en) | 2005-02-10 |
US20040248230A1 (en) | 2004-12-09 |
US20060281166A1 (en) | 2006-12-14 |
AU2002339973B2 (en) | 2007-10-18 |
JP4283111B2 (en) | 2009-06-24 |
EP1430143A2 (en) | 2004-06-23 |
CA2461019A1 (en) | 2003-04-03 |
US7094560B2 (en) | 2006-08-22 |
WO2003027238A3 (en) | 2004-02-12 |
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