PURIFICATION OF AN AMIDE FORMING ENZYME

Abstract: A method for preparing an amidating enzyme product from a solution of proteins containing the amidating enzyme comprising a heat treatment step. The solution of proteins may be derived from mammalian tissue, such as porcine pituitary gland tissue. The method may be used to enhance the specific activity of the amidating enzyme product and/or especially to purify the amidating enzyme from a solution of proteins. Optimum conditions for heat treatment are a temperature of about 60<o>C for a duration of from 5 to 10 minutes.

Description: Purification of an amide forming enzyme. Field of the Invention This invention relates to a method for preparing an enzyme product from a composition comprising other proteins. In particular, it relates to a method for preparing an amidating enzyme product of enhanced specific activity from a solution of proteins containing the amidating enzyme and especially to a method for purifying an amidating enzyme from a solution of proteins. Background to the Invention Bradbury e_t al have isolated an enzyme from porcine pituitary tissue which converts a C-terminal glycine residue of a polypeptide to an 0 -amide group (Bradbury, A.F. t l Nature, (1982), 298, 686-688; Bradbury, A.F. et a_l, Biochem. and Biophys. Comm. (1983), 112(2) 372-377; Landymore-Lin, A.E.N. e_t a_l, Biochem. and Biophys. Comm. (1983) 117(2) , 289-293). A number of physiologically active polypeptides and proteins, especially peptide hormones, have been shown to depend upon a C-terminal *&>(-amide group for their activity. The recent advances in recombinant DNA techniques have made possible the synthesis of polypeptides and proteins on an industrial scale using culture techniques. However, polypeptides and proteins produced in this manner do not possess a C-terminal amide and may not therefore exhibit their full physiological activity. This problem has been overcome by the use of an amidating enzyme to convert an additional C-terminal glycine residue to the necessary amide group (see for example, published international patent application WO 85/00043). The amidating enzyme, described by Bradury e_t a_l has, however, proved difficult to purify in sufficien quantities to make viable a commercial process based upon its its use. The purification of proteins from mammalian SUBSTITUTE SHEET tissue, in general, requires the separation of a small amount of the desired protein from a large amount of unwanted proteinaceous material. Commonly, a considerable proportion of unwanted material is removed by selective ammonium sulphate precipitation. However, this may leave a large quantity of unwanted material in the partially purified solution which may subsequently cause difficulty in the operation of high specificity purification systems such as gel filtration. Alternative methods for selective precipitation are available which employ, for example, 1 changes in pH and protein concentration. A further, but little used, method of selective precipitation employs an elevated temperature to denature and precipitate unwanted material. This method relies upon the desired protein being unusually heat stable and is not generally applicable since the majority of proteins are not stable at elevated temperature. indeed, most protein purification techniques are carried out near the freezing point of the solvent in use. We have surprisingly discovered that the mammalian amidating enzyme identified by Bradbury e_t al is unusually heat stable. This unexpected discovery allows the preparation of the enzyme by a protocol involving a heat treatment step. Summary of the Invention Accordingly, the present invention provides a method for preparing an amidating enzyme product comprising the step of heat treating a solution of proteins containing the amidating enzyme. We have discovered, in accordance with the invention that heat treatment of a solution of proteins containing the amidating enzyme gives rise to an increase in the specific activity of the enzyme. We have further discovered that heat treament may be used in the purification of amidating SUBSTITUTE SHEET enzyme from a solution containing the enzyme and other proteins such that a least some of the proteins in solution are precipitated leaving the amidating enzyme in solution. Thus, in one embodiment the invention provides a method for preparing an amidating enzyme product of enhanced specific activity comprising the step of heat treating a solution of proteins containing the amidating enzyme. The term "specific activity" as used herein is defined as ug product formed/mg total protein. The term "enhanced" as used herein denotes an increased specific activity of the amidating enzyme product relative to the starting solution of proteins containing the amidating enzyme. In a preferred embodiment, the invention provides a method for purifying an amidating enzyme from a solution of proteins, comprising the step of heat treating the solution such that at least some of the proteins in solution are precipitated, leaving amidating enzyme in solution. in a particularly preferred embodiment, the invention provides a method for purifying an amidating enzyme from a solution of proteins derived from mammalian tissue, comprising the step of heat treating the solution such that at least some of the proteins in solution are precipitated, leaving the amidating enzyme in solution. The amidating enzyme may be any thermally stable mammalian amidating enzyme, such as, for example, that found in serum and in mammalian tissues, but is preferably similar to, or identical with, the amidating enzyme found in the mammalian pituitary gland, suitably the porcine pituitary gland. . The method may involve disruption of the mammalian tissue, for example by sonication or mechanical blending in a buffer, followed, if appropriate, by centrifugation to remove insoluble material. The supernatant may then be subjected to one or more purification steps such as purification by selective precipitation with, for example, ammonium sulphate. Preferably the heat treatment step is conducted early in the purification procedure, for example, after a selective ammonium sulphate precipitation step, to substantially reduce the amount of unwanted proteinaceous material in the solution, The supernatant remaining after the heat treatment step may be further purified, by, for example, dialysis, chromatography and/or gel filtration. Conventionally, protein purification is carried out at as low a temperature as possible which, for practical purposes, means slightly above the freezing point of the solvent in use. Normally, where an aqueous buffer is used, the purification is carried out at about 4°C. As the temperature is increased, the denaturation rate of the proteins increases differentially according to the thermal stability of the proteins, under the prevailing conditions of pH and dilution. In the method of the present invention therefore the heat treatment step involves heating the solution to a suitable temperature and for a suitable period of time such that, at least, a part of the unwanted protein in the solution is denatured and precipitated, leaving the amidating enzyme in solution. The temperature used may, for example, be in the range 40°C to 80°C, more preferably in the range 50°C to 70°C and in particular about 60°C. The duration of the heat treatment step depends upon the rate of denaturation of the unwanted proteins. At 60°C a duration in the range 5 to 60 minutes may be used, advantageous results being achieved with a duration of from 5 to 10 minutes. If the duration of the heat treatment step is too great, significant denaturation of the desired amidating « enzyme may occur and a reduction in yield may arise. SUBSTITUTE SHEET Description of Embodiments The invention is now illustrated with reference to the following examples which describe the preparation of an amidating enzyme from porcine pituitary tissue. (unless otherwise stated all the steps were performed at 4°C). Example 1 1 A sample of porcine pituitary gland tissue (500g wet weight) was blended at full speed in a Waring Blender with an equal quantity of a buffer having the following composition: 50mM sodium phosphate buffer (pH 6.8), 5mM EDTA, ImM PMSF, Catalase (5Uml _1 ), 0.1% (w/v) Triton X-100, and O.lM sodium chloride. The sample was blended until it was homogenous. Insoluble debris was removed by centrifugation in a Sorvall 5B centrifuge using a 6 x 250ml head at 10,000 r.p.m. for 15 minutes. The supernatant was decanted carefully and cell debris was resuspended, reblended and centrifuged again as described above to yield a second batch of supernatant. The two batches of supernatant were pooled to give a sample having a total volume of about 1700ml. An ammonium sulphate cut (0-25% ) was performed using an ammonium sulphate quantity calculated from standard tables (Scopes R.K., "Protein Purification Principles and Practice" pub. Springer-Verlag; New York, Heidleberg, Berlin (1982)). The pellet was collected by centrifugation (as described above), the supernatant was decanted, and the ammonium sulphate concentration increased to give a 25-40% m cut. (In the precipitation stages the ammonium sulphate was added very slowly over a period of about one hour to avoid high local concentrations of ammonium sulphate). The 25-40% cut was stirred for one hour after the last addition of ammonium sulphate. The resulting solution was centrifuged as described above, and the pellet was resuspended in a solution containing 25mM piperazine-NN , -bis-2-ethanesulphonic acid monosodiu salt (PIPES pH 6.8) and ImM EDTA (conductivity •=• 2.3uS cm~^), to a final volume of about 1000ml. The resuspended pellet was divided into 100ml aliquots and placed in 250ml glass conical flasks. The flasks were each placed in a shaking water bath at about 60°C such that the level of the solution was lower than the water in the water bath. The temperature of the solution was measured with a thermometer. At 60°C the samples were incubated for 15 minutes with constant agitation. (The sample took about 5 minutes to reach 60°C). After 15 minutes the samples were removed from the water bath and cooled rapidly on ice. After about 3 minutes the temperature had dropped to about 30°C whereupon 30ul catalase (Sigma Chemicals) was added to each flask (40Uml ~ l). The samples were cooled to about 5°C before centrifugation (as described above) to remove denatured protein. The supernatant was carefully decanted (total volume about 800ml) and the conductivity was measured (conductivity = 44 iiS cm ~ l). The conductivity of the supernatant was decreased by dilution with PIPES buffer as described above, followed by concentration using an Amicon ultrafiltration cell. The conductivity was decreased to 5.4 us cm ~ before the volume was concentrated to about 800ml. A column (9.0 x 26.0cm) of DEAE-Cellulose (DE-52) was prepared and equilibrated with 25mM PIPES (pH 6.8) and ImM EDTA. The total bed volume was 1653ml, the flow rate was 560ml hr ~ l and fractions of 65ml were collected. The sample was applied to the column, which was then washed wi_i_ about 4 litres of buffer. A linear gradient of 0-500mM sodium chloride was then applied in the equilibrating buffer at a total volume of 4000ml. The conductivity was measured to indicate the true gradient and activity monitored to detect active fractions. An. active fraction (active amidating enzyme) was eluted at about 60mM sodium chloride as SUBSTITUTE SHEET the first peak to come off the column after the gradient was applied. Active fractions were pooled and concentrated under nitrogen using a 200ml A icon ultrafiltration cell containing a PM 10 membrane (10000 m.w. cut-off) to a final volume of 40ml. The concentrated sample was applied, at room temperature, to a column (4.4 x 75cm) of Ultragel AcA 54 equilibrated with 25mM PIPES (pH 6.8) and 0.4M sodium chloride. The total bed volume was 1140ml, the flow rate was 44ml h ~l and fractions of 11ml were collected and activity measured. Active fractions eluted on the trailing side of the second protein peak from the column (peak activity about 495-572ml). The active fractions were stored separately at -20°C. The yield of the purification method was considerably greater than the yield obtained using an analogous method without a heat treatment step. Example 2 A 500g (wet weight) sample of porcine pituitary tissue was blended at full speed in a waring blender with 500mls of a buffer of the following composition: 50mM PIPES buffer (pH 6.8), lOOuM NaCl, 0.1% (w/v) Triton X-100, Catalase (lOOu/ l), ImM PMSF. The sample was blended until it was homogeneous. The centrifugation and subsequent reblending procedure was as described in Example 1. From the supernatant obtained, 100ml aliquots were taken and placed in 250ml glass conical flasks. These were subjected to heat treatment at 60°C. The procedure was as* described in Example 1. Samples were removed after 5, 10, 15, 30 and 60 minutes and treated as described in Example 1. The C-^-amidating enzyme activity in each sample was determined and compared with the enzyme activity in a supernatant not subjected to heat treatment. The activity of the sample not subjected to heat treatment was assumed to be 100%. The protein concentration of the sample was measured using the Pierce BCA Protein Assay Reagent (supplied by Pierce Ltd. product No.23225). Duration of heat Protein Activity treatment at 60°C concentration (%) recovered (%) (Mins) 0 100% 100% 5 t 39% 149% 10 35% 119% 15 35% 149% 30 32% 99% 60 30% 62% The above table indicates that there is a significant reduction in the total protein concentration after 5 minutes at 60°C, but the enzyme activity shows a significant increase between 5 and 15 minutes . At 60 minutes there remains a significant enzyme activity. The >«(-amidating enzyme may be further purified as described in the previous example or by using standard protein purification techniques. It will of course be understood that the present invention has been described above purely by way of example, and modification of details can be made within the scope and spirit of the invention. SUBSTITUTE SHEET

Claims: CLAIMS; 1. A method for preparing an amidating enzyme product comprising the step of heat treating a solution of proteins containing the amidating enzyme. 2. A method for preparing an amidating enzyme product of enhanced specific activity comprising the step of heat treating a solution of proteins containing the amidating enzyme. 3. A method for purifying an amidating enzyme from a solution of proteins comprising the step of heat treating the solution such that at least some of the proteins in solution are precipitated, leaving the amidating enzyme in solution. 4. A method according to any one of claims 1 to 3 wherein the solution is derived from mammalian tissue. 5. A method according to claim 4 wherein the mammalian tissue is a mammalian pituitary gland. 6. A method according to claims any one of 1 to 3 wherein the step of heat treating the solution is conducted at about 60°C. 7. A method according to claim 6 wherein the step of heat treating the solution is of 5 to 10 minutes duration. 8. An amidating enzyme product when prepared by a method according to any one of claims 1 to 7.

Inventors: EATON MICHAEL ANTHONY WILLIAM, LAWTON STEPHEN ASHLEY, EATON, MICHAEL, ANTHONY, WILLIAM, LAWTON, STEPHEN, ASHLEY
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1986
Main classification: C12N9
IPC codes: C12N9