A method for activating epidermal growth factor (EGF)

Abstract: The present invention relates to a method for activating protein wherein protein produced in a biologically inactive form (non-natural-form protein) is converted into a biologically active protein (natural-form protein) by bringing it into contact with cultured cells of an organism, and according to the present invention, the non-natural-form protein can be converted efficiently into the natural-form protein having activity, so the yield of the natural-form protein can be further raised by subjecting, e.g., culture of transformant to the activation treatment.

Description: DETAILED DESCRIPTION OF THE INVENTION: Technical Field to which the Invention Belongs The present invention relates to a method for converting isolated epidermal growth factor (EGF) which for some reasons, is rendered biologically inactive thus failing to have a biological activity which it would have in its normal stereostructure, into the biologically active EGF. More specifically, the present invention relates to a method for activating EGF produced in transformant by means of genetic engineering and rendered biologically inactive due to formation of a stereostructure different from that of its natural EGF. Prior Art Enzymes derived from organisms, peptides and proteins having a biological activity such as cytokine, are produced in a large amount by use of microorganisms, animal and plant cultured cells. However, it is known that heterologous proteins expressed in microorganisms as hosts often undergo denaturation in the host cells and occur in biologically inactive forms as precipitates. Further, even if produced and secreted extracellularly, the presence of peptides or proteins in biologically inactive forms is also known. One speculative reason is that although the proteins have the same primary structures as those of their natural types, their stereostructures are biologically not correctly constituted, thus failing to attain structures having biological activities. Further, it is known that chemically synthesized polypeptides and biosynthesized proteins in cell extracts (cell-free system) often fail to have correct stereostructures, and these have, e.g., inactive structures with relatively less folding lacking in disulfide linkages. JP-A 59-161321 discloses a method for restoring the activity of protein in a biologically inactive state failing to attain a correct stereostructure owing to incomplete disulfide linkages, wherein precipitated inactive protein is solubilized with a strong detergent, then the solubilized fluid is diluted or the strong detergent is replaced by a weak detergent thereby making the inactive protein active, and the biologically active protein is then recovered. However, this prior art method suffers from the problems that the volume of the protein solution is significantly increased and the activation is time-consuming. Miyauchi et al. (Journal of Industrial Microbiology & Biotechnologie, Vol. 21, pp. 208-214 (1998)) discloses a method for producing the native form of recombinant epidermal growth factor (EGF) using expanded bed adsorption. No satisfactory method has been developed. Problems to be Solved by the Invention To raise the yield of EGF produced by recombinant DNA technology, it is conventional to take approaches for improving a gene, e.g., for modification of a signal peptide or for improving a method of culturing a transformant. Contrary to the prior art, the present application is drawn to an indirect method for raising the yield of target EGF not by directly raising the yield thereof, but by converting inactive EGF produced in a culture into an active type, and the object of the present invention is to provide a method for activating isolated EGF not having a biological activity for reason of failing to have an active stereostructure or for any other reasons, so that EGF can be converted into the biologically active EGF very easily and rapidly as compared with the previously proposed or practiced methods. Further, the present invention provides a method for separating and fractionating EGF produced in a biologically inactive form from a solution containing both EGF produced in a biologically active form and the EGF produced in a biologically inactive form, wherein the EGF produced in a biologically inactive form resulting from, e.g., incorrect disulfide linkages is specifically aggregated, precipitated and recovered. As a matter of course, the inactive EGF separated and fractionated in this manner can also be activated by the method described above. Brief Description of the Drawings Fig. 1 is a graph showing the conversion of inactive EGF into active EGF (natural-form) by the cells of Brevibacillus choshinensis . Fig. 2 is a graph showing the conversion of inactive EGF into active EGF (natural-form) by the cells of Bacillus subtilis . Fig. 3 is a graph showing the conversion of inactive EGF into active EGF (natural-form) in a reactor having the cells of E . coli immobilized therein. Fig. 4 is a graph showing the conversion of inactive EGF into active EGF (natural-form) by the cells of yeast. Means for Solving the Problems The present invention was made to achieve the object described above, and as a result of eager study, the present inventors first found that when isolated multimeric EGF not having a biological activity for reason of failing to achieve a normal stereostructure is incubated in a suspension of cultured cells of bacteria of the genus Brevibacillus , its stereostructure is normalized and its original biological activity is thereby restored, and the multimeric EGF is thus converted into active EGF (native-form). As a result of additional examination, the present invention was thereby completed. That is, the present invention relates to a method for activating a biologically-inactive form of epidermal growth factor (EGF) produced by recombinant DNA technology, of which the inactivity is due to erroneous disulfide linkages, which comprises bringing said inactive form of EGF, wherein the EGF is an isolated protein, into contact with cultured microbial cells in an aqueous solution. Hereinafter, the present invention is described in detail. (1) The cultured cells of organisms used in the present invention are microbial cells. The microbial cells used in the present invention may be prokaryotic cells, such as bacteria of the genus Escherichia , bacteria of the genus Bacillus , bacteria of Brevibacillus etc., or may be eukaryotic cells, such as yeasts of the genus Saccharomyces , yeasts of the genus Pichia, fungi of the genus Aspergillus etc. The cultured cells described above can be used as necessary, and particularly for industrial purposes, the cells are preferably suitable for large-scale culture, more preferably cells of fungi, yeasts and bacteria which are cultured preferably under aerobic conditions. In particular, the genera Escherichia, Bacillus , Brevibacillus and yeasts whose industrial and large-scale production has been established can be used more preferably. Among bacteria of the genus Brevibaccillus , Brevibaccillus choshinensis HPD31 does not produce a proteolytic enzyme in a culture. This strain was deposited as Bacillus brevis H102, FERM BP-1087 on June 24, 1986 and renamed as Brevibacillus choshinensis HPD31, FERM BP-1087 and the deposition runs. However, many years have passed since the date of the deposition, and therefore this strain was newly deposited on August 31, 1999 under the Budapest Treaty in the same depository and alloted No. FERM BP-6863. Depository: National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology. Further, this strain is the same strain as Bacillus brevis HPD31. Its position in classification was altered in International Journal of Systematic Bacteriology, Vol. 46, pp. 939-946 (1996)), and by use of this bacterium, EGF having no biological activity is converted into activated EGF having a normal stereostructure and this EGF is accumulated without undergoing decomposition during reaction, thus permitting the process of the present invention to be carried out very efficiently. Further, because no proteolytic enzyme occurs in the medium, the culture or the cells collected by centrifugation can be used advantageously as such without worrying about decomposition of the activated EGF. (2) The culture method varies depending on the genus and species of cells used, but any medium may be used insofar as the cells can be cultured and proliferated therein in a conventional culture method. For example, it is possible to use cultured cells harvested after proliferation by a culture method such as a liquid shake culture method, an agar plate culture method, a solid culture method, an aeration stirring culture method etc. The cells thus cultured may be used in the form of culture itself containing the cells, or the cells may be used as such after collection by centrifugation or by filtration of the culture through an MF membrane ( microfiltration membrane ), or may be used after washing with various buffers. The collected cells may be lyophilized and suspended in a buffer before use, or may be suspended in a buffer for freezing storage, frozen for storage, and thawed before use. As described above, the cultured cells used in the present invention may be used in at least one form selected from the group consisting of culture, cells harvested by centrifugation etc., lyophilized cells, cells in the form of suspension of lyophilized cells in a suitable buffer, any suspended cells after cultured cells were lyophilized and thawed; but in any case, they should be used as undisrupted (undestroyed) cells. (3) EGF The EGF produced by recombinant DNA technology include (1) EGF itself, and (2) biologically inactive EGF separated and fractionated from the culture. Some recombinant proteins secreted into the outside of the cells are insolubilized in the outside of the cells, or fail to attain their correct structures, thus being aggregated and produced in a multimeric state, depending on their type. Usually, such proteins have incorrectly bridged disulfide linkages to form incorrect linkages. Therefor, these proteins, though having the correct primary structure, have different stereostructures different from those of the active types, thus failing to have their biological activities. The present invention relates to a method-of converting isolated EGF into a biologically active EGF by bringing it into contact with cultured cells in order to reconstitute disulfide linkages into correct ones, that is, a method of activating isolated EGF produced in a biologically inactive state. (4) To carry out the present invention, the EGF to be activated is brought into contact with cultured cells. Bringing the EGF produced in a biologically inactive state into contact with cultured cells of organism involves bringing it into contact with at least one member selected from the group consisting of cultured cells themselves, culture containing cultured cells, washed cultured-cells, immobilized cultured-cells, lyophilized cultured-cells, and a suspension of at least one member selected from the group consisting of them. Although the mechanism of the activation according to the present invention has not been elucidated, one speculative reason is that disulfide linkages are normalized. In the method of activating EGF according to the present invention, the best embodiments can be selected for the contact manner and the amount of cultured cells to be contacted with EGF produced in a biologically inactive state. For example, if microorganisms having prokaryotic cells are used as the cultured cells, the microbial cells are suspended in a suitable buffer, e.g., Tris-HCl buffer, and the suspension is added in excess to EGF produced in an inactive state, mixed well, incubated for 1 to 2 hours preferably at 15 to 60 °C, more preferably at 25 to 45 °C, whereby the EGF produced in a biologically inactive state is activated. For the contact, the cultured cells are immobilized on a MF membrane (microfiltration membrane), and a EGF solution prepared by dissolving in buffer is brought into contact with the cells by passing it through the membrane, or alternatively the cells are adsorbed on a carrier such as hydrophobic porous resin HP-20 (Mitsubishi Chemicals Co.) to prepare an immobilization column, and the EGF solution may be brought into contact with the cells by feeding it to the immobilization column. The activation apparatus which can be used in the method of the present invention is to realize these steps specifically, and this apparatus includes cultured cells immobilized on an MF membrane or insoluble carrier, and a solution feeder such as a pump by which isolated EGF produced in a biologically inactive state is fed to in the form of solution and brought into contact with the immobilized cells, thereby activating EGF. In the apparatus, the cultured cells may be immobilized by conventional techniques of immobilization onto carriers for enzymes, various proteins and microorganisms, besides the means described above. That is, inorganic or organic, natural or synthetic, high-molecular or low-molecular, biological or non-biological carriers may be used, and conventionally known immobilization techniques such as covalent bonding method, ionic bonding method, physical adsorption method are utilizable as necessary. As the carrier, are suitably usable polysaccharide (derivative) such as agarose, cellulose, dextran, Sepharose, Sephadex, synthetic high molecular material such as polyacrylamide gel, polystyrene, and inorganic or ceramic material such as glass (e.g., porous glass), ceramic (e.g., porous ceramic), activated carbon, silica gel, alumina, clay minerals may be used. Immobilization may be carried out by bringing the cultured cells into contact with the carrier, or by bringing the cultured cells into contact with the carrier after the carrier is reacted with cyan bromide or cyanoboron hydride. Or the cultured cells may be more strongly immobilized on the carrier after the carrier is treated with a cross-linking agent or a condensing agent. The effect of the present invention can be improved more effectively by adding 0.1 to 50 mM compound having sulfhydryl group(s), such as reduced glutathione and cysteme, when the cultured cells are brought into contact with the isolated EGF which was produced in a biologically inactive form due to erroneous disulfide linkages. Further, in the case of a mixture of natural-form EGF and non-natural-form EGF from which the EGF to be activated is not isolated, the present invention provides a method for separating and fractionating the EGF produced in a biologically inactive form, wherein a solution containing EGF produced in a biologically active form and EGF produced in an inactive form is adjusted to a pH value less than the isoelectric point of the biologically active form, whereby the EGF produced in a biologically inactive form is specifically aggregated, precipitated and recovered. According to this method, EGF can be purified, and the recovered inactive EGF can also be used as the EGF to be activated. In one example of the fractionation and separation method of the present invention, a solution containing recombinant epidermal growth factor produced in a biologically inactive form due to its erroneous disulfide linkages is adjusted to pH 3 whereby the recombinant epidermal growth factor as the protein produced in a biologically inactive form is specifically aggregated, precipitated and recovered. Hereinafter, the present invention is described in more detail by reference to the Examples. Example 1: Preparation of cultured cells (1) Preparation of E . coli cells Escherichia coli JM109 (Takara Shuzo Co., Ltd.) was cultured at 30 °C for 24 hours with shaking in a liquid medium containing 4 g polypeptone, 0.5 g yeast extract, 2 g glucose and 100 ml water (pH 7.0) and harvested by centrifugation, and the cells thus obtained were suspended in 100 ml of an aqueous 4 % polypeptone solution. (2) Preparation of Bacillus subtilis cells Bacillus subtilis IFO13719 was cultured at 30 °C for 24 hours with shaking in a liquid medium containing 4 g polypeptone, 0.5 g yeast extract, 2 g glucose and 100 ml water (pH 7.0) and harvested by centrifugation, and the cells thus obtained were suspended in 100 ml of an aqueous 4 % polypeptone solution. (3) Preparation of Brevibacillus choshinensis cells Brevibacillus choshinensis HPD31 (FERM BP-1087) was cultured at 30 °C for 24 hours with shaking in a liquid medium containing 4 g polypeptone, 0.5 g yeast extract, 2 g glucose and 100 ml water (pH 7.0) and harvested by centrifugation, and the cells thus obtained were suspended in 100 ml of an aqueous 4 % polypeptone solution. (4) Preparation of yeast cells Saccharomyces cerevisiae IFO10217 was cultured at 30 °C for 24 hours with shaking in a liquid medium containing 1 g peptone, 0.5 g yeast extract, 4 g glucose, 0.5 g KH 2 PO 4 , 0.2 g MgSO 4 ·7H 2 O, and 100 ml water (pH 7.0) and harvested by centrifugation, and the cells thus obtained were suspended in 100 ml of an aqueous 1 % peptone solution. Example 2: Method of separating and fractionating protein produced in an inactive form (1) Preparation of human recombinant epidermal growth factor (h-rEGF) produced in an inactive form Brevibacillus choshinensis HPD31 carrying plasmid pHT110EGF containing a gene for human recombinant epidermal growth factor, prepared by Miyauchi et al. (Journal of Industrial Microbiology and Biotechnology, Vol. 21, pages 208-214 (1998)), was cultured in a liquid medium for 65 hours under the conditions described in the same literature, and the supernatant was recovered by centrifugation. It was adjusted to pH 3.0 with 6 M hydrochloric acid, and the human recombinant epidermal growth factor produced in an inactive form was specifically precipitated, and the resulting precipitate was recovered by centrifugation. The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.0) and adjusted again to pH 3.0 with 6 M hydrochloric acid, and the precipitate was recovered by centrifugation. Further, the precipitate was suspended in distilled water, adjusted to pH 7.0 with aqueous sodium hydroxide and lyophilized to prepare inactive human recombinant epidermal growth factor (h-rEGF). This inactivated human recombinant epidermal growth factor (h-rEGF) is multimeric EGF (according to SDS-PAGE/Western Blotting analysis of the culture supernatant producing EGF), and it is a non-natural-form (non-native-form) EGF whose inactivation is presumably due to intermolecular formation of S-S linkages between cysteine residues of neighboring EGF molecules. Example 3: Activation of inactivated proteins (1) Activation of inactive human recombinant epidermal growth factor (h-rEGF) by the cultured cells of Brevibacillus choshinensis The inactive h-rEGF obtained in Example 2-(1) was dissolved in distilled water to give a solution (dried matter 36 mg/ml). To 20 µl of the solution, were added (a) 20 µl of 1 M Tris-HCl buffer (pH 8.0), (b) 4 µl of 100 mM reduced glutathione, (c) 5 µl, 20 µl or 50 µl of a suspension prepared by diluting the suspension of Brevibacillus choshinensis obtained in Example 1-(3) with an aqueous 4 % polypeptone solution so that the diluted suspension could show O.D. 10 at 660 nm, and (d) distilled water to give a mixed solution of 200 µl (total volume). The mixed solution was incubated at 30 °C, and the conversion of the inactive multimeric EGF into active natural-form EGF was examined with time. The conversion of the multimeric EGF into natural-form EGF was quantified by HPLC. The conditions for HPLC analysis were conducted using Licrospher 100, RP-18 column (4x125 mm) (Merck Co.), and the protein was eluted by a gradient of from 25 % to 34 % acetonitrile containing 0.1 % trifluoroacetic acid. The flow rate was 1.0 ml/min and the protein was detected at 280 nm. The increase of the area in a peak appearing at the retention time of standard hEGF was regarded as the amount of natural-form EGF converted. The conversion of the multimeric EGF into natural-form EGF is shown in the relationship between time (min, abscissa) and the amount of natural-form EGF produced (mg/ml, ordinate) in Fig. 1. The results of addition of 50 µl (▴), 20 µl (Δ) and 5 µl () of the Brevibacillus choshinensis cell suspension and the control (○) are shown. The control was prepared in the same manner as in the preparation of the mixed solution, provided that 50 µl of an aqueous 4 % polypeptine solution was used in place of (c). When the Brevibacillus choshinensis cell suspension was added, the appearance of the natural-form EGF was promoted, and this conversion was increased as the density of the Brevibacillus choshinensis cells was increased. No addition of the suspension means the use of the polypeptone solution without containing the cells. This is also applied in the following (2) to (4). (2) Activation of inactive human recombinant epidermal growth factor (h-rEGF) by the cultured cells of Bacillus subtilis The inactive h-rEGF obtained in Example 2-(1) was dissolved in distilled water to give a solution (dried matter 36 mg/ml). To 20 µl of the solution, were added (a) 20 µl of 1 M Tris-HCl buffer (pH 8.0), (b) 4 µl of 100 mM reduced glutathione, (c) 50 µl of a suspension prepared by diluting the suspension of Bacillus subtilis obtained in Example 1-(2) with an aqueous 4 % polypeptone solution so that the diluted suspension could show O.D. 10 at 660 nm, and (d) distilled water to give a mixed solution of 200 µl (total volume). The mixed solution was incubated at 30°C, and the conversion of the inactive EGF into active natural-form EGF was examined with time. The conversion of the multimeric EGF into natural-form EGF was quantified by the same manner as in Example 3-(1). The conversion of the multimeric EGF into natural-form EGF is shown in the relationship between time (min, abscissa) and the amount of natural-form EGF produced (mg/ml, ordinate). The results of addition (▴) of the Bacillus subtilis cell suspension and the control (○) are shown. The control was prepared in the same manner as in the preparation of the mixed solution, provided that 50 µl of an aqueous 4 % polypeptone solution was used in place of (c). When the Bacillus subtilis cell suspension was added, the appearance of natural-form EGF was promoted. (3) Activation of inactive human recombinant epidermal growth factor (h-rEGF) by the cultured cells of E . coli A tangential flow-type filter Minitan II (Millipore Co.) equipped with a MF membrane (Durapore (trade name), Millipore Co.) with pores, of which the diameters are 0.45 µm, was used as an immobilized cell reactor. The inactive h-rEGF obtained in Example 2-(1) was dissolved in distilled water to give a solution (dried matter 36 mg/ml). To 100 ml of the solution, were added (a) 100 ml of 1 M Tris-HCl buffer (pH 8.0), (b) 50 ml of a suspension prepared by diluting the suspension of E . coli obtained in Example 1-(1) with an aqueous 4 % polypeptone solution so that the diluted suspension could show O.D. 10 at 660 nm, (c) 20 ml of 100 mM reduced glutathione, and (d) 730 ml of distilled water to give a mixed solution of 1000 ml (total volume). The immobilized cell reactor installed in a thermostatic chamber set at 30°C, was fed with the mixed solution at a flow rate of 10 ml/min from a server tank containing the mixed solution of 1000 ml by using a pump. The flow rate of the filtrate passed through the MF membrane was regulated to attain 1 ml/min by controlling the pressure at the outlet valve of the immobilized cell reactor; the filtrate contains the activated h-rEGF. A polypeptone-containing solution, which was prepared in the same manner as in the preparation of the mixed solution provided that 50 ml of an aqueous 4 % polypeptone solution was used in place of (b), was fed to the server tank at the same flow rate as that of the filtrate (i.e., 1 ml/min) to prevent the decrease of the volume of the solution in the immobilized cell reactor. The filtrate was recovered with time, and the conversion of the multimeric EGF into natural-form EGF was quantified in the same manner as in Example 3-(1). The change with time of conversion of the multimeric EGF into natural-form EGF is shown in the relationship between time (min, abscissa) and the-amount of natural-form EGF produced (mg/ml, ordinate) in Fig. 3. In the case (▴) where the E . coli cell suspension was added at the start of the reaction, the conversion into natural-form EGF was promoted as compared with the case of the control (○), and further, the natural-form EGF was successively recovered for a long period of time. The control was prepared in the same manner as in the preparation of the mixed solution, provided that 50 ml of an aqueous 4 % polypeptone solution was used in place of (b). (4) Activation of inactive human recombinant epidermal growth factor (h-rEGF) by the cultured cells of yeast The inactive h-rEGF obtained in Example 2-(1) was dissolved in distilled water to give a solution (dried matter 36 mg/ml). To 20 µl of the solution, were added (a) 20 µl of 1 M Tris-HCl (pH 8.0), (b) 4 µl of 100 mM reduced glutathione, (c) 50 µl of'a suspension prepared by diluting the suspension of S . cerevisiae obtained in Example 1-(4) with an aqueous 1 % polypeptone solution so that the diluted suspension could show O.D. 10 at 660 nm, and (d) distilled water to give a mixed solution of 200 µl (total volume). The mixed solution was incubated at 30 °C, and the conversion of the inactive EGF into active natural-form EGF was examined with time. The amount of natural-form EGF converted from the inactive EGF was examined in the method shown in Example 3-(1). The change with time of conversion from the multimeric EGF into natural-form EGF is shown in the relationship between time (min, abscissa) and the amount of natural-form EGF produced (mg/ml, ordinate) in Fig. 4. The results of addition (▴) of the yeast cell suspension and the control (○) are shown. The control was prepared in the same manner as in the preparation of the mixed solution, provided that 50 µl of an aqueous 1 % polypeptone solution was used in place of (c). When the yeast cell suspension was added, the appearance of natural-form EGF was promoted. Effects of the Invention According to the present invention, inactive EGF can be converted into active EGF in the very easy treatment of bringing the isolated EGF into contact with cultured cells. Therefore, according to the activation method of the present invention, EGF (often containing inactive EGF, thus reducing the yield of active EGF and further leading in some cases to no recovery) produced by recombinant DNA technology can be activated by incubating the produced culture containing inactive EGF with culture cells, and as a result, the yield of natural-form EGF having activity can be raised.

Claims: A method for activating a biologically-inactive form of epidermal growth factor (EGF) produced by recombinant DNA technology, of which the inactivity is due to erroneous disulfide linkages, which comprises bringing said inactive form of EGF, wherein the EGF is an isolated protein, into contact with cultured microbial cells in an aqueous solution. The method according to claim 1, wherein the cultured microbial cells are cultured cells of at least one member selected from the group consisting of prokaryotes and eukaryotes. The method according to any of claims 1 or 2, wherein the cultured microbial cells are cultured cells immobilized on a microfiltration membrane or a column containing an insoluble carrier. The method according to any of claims 1 to 3, wherein the biologically inactive form of the EGF is recovered by adjusting a solution containing the biologically active form of the EGF as well as the biologically inactive form of the EGF to a pH value which is less than the iso-electric point of the biologically active form of EGF, thereby aggregating, precipitating and recovering the biologically inactive form of EGF.

Inventors: MIYAUCHI AKIRA, MIZUKAMI MAKOTO, OZAWA MAKOTO, YOSHIDA MASATO, MIYAUCHI, AKIRA, MIZUKAMI, MAKOTO, OZAWA, MAKOTO, YOSHIDA, MASATO
Applicants: HIGETA SHOYU KK, HIGETA SHOYU CO., LTD.
Application year: 1999
Main classification: C07K1
IPC codes: C12N15, C07K1, C12N1, C07K14