AU767225B2 - A method for the manufacture of protein having biological activity - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

.*TO BE COMPLETED BY APPLICANT Name of Applicant: HIGETA SHOYU CO., LTD.

Actual Inventors: AKIRA MIYAUCHI, MAKOTO OZAWA, MASATO YOSHIDA, MAKOTO MIZUKAMI, HIROAKI TAKAGI Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: A METHOD FOR THE MANUFACTURE OF PROTEIN HAVING BIOLOGICAL ACTIVITY The following statement is a full description of this invention, including the best method of performing it known to me:- performing it known to me:- 06/04/00,mcl 040.cs,l A METHOD FOR THE MANUFACTURE OF PROTEIN HAVING BIOLOGICAL

ACTIVITY

DETAILED DESCRIPTION OF THE INVENTION: Technical Field to which the Invention Belongs The present invention relates to a method for the manufacture of protein having biological activity, wherein, when a protein, of which the native-form protein has a normal biological activity, is in a non-native-form state which is biologically inactive due to the reason that its stereostructure is a high-order structure which is biologically different from the stereostructure of the native-form protein or for other reasons, the protein produced in a biologically inactive state is converted into an active native-form protein.

The present invention relates to a method for the manufacture of protein wherein a protein, which is biologically S active if produced, for example, in a normal stereostructure, but is produced in a biologically inactive state due to some reasons, is converted into a biologically active protein and recovered. More particularly, it relates to a method for the manufacture of protein which is characterized in that, when a protein produced by a transformed microorganism prepared, for example, by means of genetic engineering may has stereostructure different from that of the native-form protein and the protein may be in a biologically inactive state, the addition of a sulfhydryl compound in the incubation of the transformed microorganism changes the inactive state into the active state and the active protein is recovered.

Prior Art It has now been possible to manufacture a large amount of active peptides and proteins derived from organisms such as enzymes and cytokines by use of microorganisms and cultured cells of animals and plants by means of genetic engineering.

However, it is known that exogenous proteins expressed by use of bacterium or the like as a host are often denatured in the host cells and are present in a biologically inactive state as an inclusion body, and also known that there are peptides and proteins which are in a biologically inactive state even if *i secreted and produced extracellularly. One of the reasons therefor is believed to be that, although the expressed proteins .have the same correct primary structures as those of the native-form proteins, the stereostructures are not correctly constructed biologically whereby the resulting structures have no biological activity.

It is further known that chemically synthesized polypeptides and biologically synthesized proteins in cell extracts (cell-free system) often fail to have correct stereostructures, and that they have, for example, inactive structures with relatively less foldings lacking in disufide bonds.

However, the current status is that there is no common method for activating the inactive proteins, that the only means is to find out an activating condition for each particular protein after trial and error, that much endeavor is needed, and that satisfactory results are neverthless not so often achieved.

Problems to be Solved by the Invention In order to obtain, in a large quantity, protein which is produced only in a very small amount in vivo, a recombinant DNA technology has been developed, for example, and, for further increase of the yield, improvement in gene itself such as modification of promoter and signal peptide or S improvement in a method for the culture of transformant has been carried out. On the contrary to the above, the present .inventors have set a technical matter for developing the so-called indirect method wherein the yield of the aimed protein is not directly increased as such but it is increased by converting the inactive protein produced in the culture products into the active protein, by paying their attention to the fact that, in the culture products, protein exists abundantly-but it is mostly inactive.

An object of the present invention is to provide a method for the activation of protein which has no biological activity or is inactivated for various reasons including the deficiency of active stereostructure. As compared with the methods which have been proposed or carried out, in the method of the present invention the conversion of such protein into biologically active protein can be carried out very easily and within a short period of time.

Brief Description of the Drawings SFig. 1 is a graph showing the changes in production amount of active human epidermal growth factor (EGF) by addition of reduced glutathione to a liquid medium of recombinant Brevibacillus choshinensis.

Means for Solving the Problems The present invention has been carried out for achieving the above-mentioned object. After an intensive investigation, the present inventors have found a method for the manufacture of protein wherein, when a sulfhydryl compound is added, during an incubating step, to a biologically inactive protein which is produced in a different stereostructure from that of native protein in incubation of a transformant, the inactive protein can be converted into a biologically active one and recovered therefrom. As a result of further investigation, the present inventors have at last accomplished the present invention.

Thus, the present invention relates to a method for the manufacture of protein, characterized in that protein which is produced in a biologically inactive state is converted into a biologically active protein by bringing it into contact with a sulfhydryl compound whereby an active protein is efficiently manufactured. In other words, the present invention newly provides a very efficient and industrial method for converting non-nativeform proteins into native-form proteins, in which all non-native-form proteins having no biological activity due to various reasons such as a biologically incorrect stereostructure, irrespective of detail mechanisms thereof, are activated by such a very simple operation that brings about said contact with a sulfhydryl compound.

In one aspect the present invention provides a method for the manufacture of a biologically active protein, characterized in that a transformant is incubated such that the transformant secretes an exogenous protein into the incubation medium, wherein said exogenous protein, when secreted from said transformant is biologically inactive, and wherein a sulfhydryl compound is added to the medium which when contacted with said biologically inactive protein converts the biologically inactive protein to a biologically active protein, and wherein said biologically active protein is recovered from the medium.

The sulfhydryl compound may be added at any time during the incubation at initiation "of incubation or at a later stage).

The present invention will now be further illustrated as follows.

20 Various examples for extracellular secretion and production of proteins by incubation of transformants have been reported. Examples of the microorganism utilized in the present invention include prokaryotic microorganisms such as bacteria of genera Escherichia, Bacillus and Brevibacillus and eucaryotic microorganisms such as yeasts of genera Saccharomyces and Pichia and fungi of genus Aspergillus. Especially in an industrial production, cells which can be incubated in large quantities are preferred. With regard to an example satisfying such a condition, an incubating condition under aerobic condition is preferred and cells of filamentous fungi, yeasts and bacteria are more preferred *o o *oooo 05/09/03,sw 1040spa.doc,5 -6examples. Genera Escherichia, Bacillus and Brevibacillus and yeasts which have been established for their industrially large-scale incubation are particularly preferably applied.

Among the above genus Brevibacillus, it is possible to secrete and produce the protein efficiently by use of cells of Brevibacillus choshinensis HPD31 (incidentally, this strain was deposited on June 24, 1986 as H102, FERM BP-108 and changed its name to Brevibacillus choshinensis HPD31 and its deposition has been still continuing. However, since several years passed since the above deposition date, this strain has been newly deposited on August 31,1999 under the Budapest Treaty and assigned number FERM BP- 6863. International Patent Organism Depository, Name of the depository institution is the National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan. This strain is the same strain as Bacillus brevis HPD31. Its taxonomic position has been changed in International Journal of Systematic Bacteriology, volume 46, pages 939-946 (1996)).

Examples of the protein which is produced by the transformant to which the present invention is applied include enzymes, hormones, cytokines, lymphokines, receptors, etc., and more specific examples include urokinase, insulin, tissue plasminogen activator, erythropoietin, interleukin receptors and the like. Examples of growth factor and of e* protein similar .*00 0* oo o o *oe *o• 05/09/03,sw 1040spa.doc,6 to growth factor include epidermal growth factor, insulin-like growth factor, immunoglobulin, single-stranded antibody, etc.

In an example of biologically inactive protein, the protein which is extracellularly secreted and produced is insolubilized after having been secreted outside of the cells, or the protein is unable to constitute the inherent stereostructure whereby protein molecules are associated each other and produced in a high-molecular state, etc., depending upon the incubating conditions, host strains, type of the produced protein, etc.

Usually, in such proteins, disulfide bond is not normally linked but is wrongly cross-linked and, therefore, although the primary structure of the protein is the same, it shows no biological activity due to difference in the stereostructure.

The ratio of production of such a biologically inactive protein .to that of normal protein having biological activity varies depending upon various conditions and, at present, no definite law or the like has been clarified yet.

According to the present invention, a sulfhydryl compound is used whereby the above-mentioned protein having no biological activity (inactive protein) is efficiently activated. In the case of secretion and production of protein by an industrial incubation of the above-mentioned transformant, it is a serious problem that a part of the aimed protein is not biologically active even if the total amount of the protein produced thereby is large. According to the present invention, however, it is possible for the disulfide bond of the secreted and produced protein to have a correct structure during the course of incubation and, as a result, the above-mentioned problem is solved and the present invention achieves a very desirable effect in terms of manufacturing efficiency.

In accordance with the present invention, it is possible that, by addition of a sulfhydryl compound to the incubating step in the production of the aimed protein by incubation of a transformant, the inactive protein is activated to give a desired protein (native-form protein having activity) in a large amount. For example, when a sulfhydryl compound is added during the incubation of Brevibacillus choshinensis HPD31 having a recombinant human epideraml growth factor gene, the production of hEGF of a native-form structure having biological activity during the incubation can be significantly increased.

With regard to such a sulfhydryl compound, any compound having a sulfhydryl group may be used and reduced glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, etc.

are used particularly effectively. The concentration of the sulfhydryl compound to be added is preferably 0.01 mM to M or, more preferably, 0.1 to 50 mM. With regard to the timing of adding it to the incubation medium, the compound may be added at any time between initiation and completion of the incubation but addition after a logarithmic growth phase in the incubation is more effective. With regard to the pH during the incubation, any pH will do so far as the microorganism is capable of growing but the range of pH of from 5 to 10 is particularly preferred.

With regard to the temperature during the incubation, any temperature will do so far as the microorganism is capable of growing but, when the incubation is carried out preferably at from 15 to 60 0 C or, more preferably, at from 25 to 45 0

C,

:...production of active protein increases.

The present invention has been illustrated 0*@e hereinabove by taking protein produced by incubation of transformant as an example of inactive protein although, besides that, various polypeptides chemically synthesized by a peptide synthesizer, various proteins biologically synthesized in a cell extract (cell free type) such as an in vitro protein synthetic system using reticulocyte extract of rabbits, various proteins produced by means of recombinant DNA technology, and a substance containing it/them may be exemplified.

In addition to the protein itself produced by incubation of transformant as mentioned above, the whole culture medium containing incubated trans formant, biologically inactive protein which is fractionated and separated therefrom, a substance containing it/them, etc. are exemplified as the protein produced by means of a recombinant DNA technology.

In carrying out the present invention, it is a good enough that the above-mentioned inactive protein (protein which is to be activated) is made to contact with a sulfhydryl compound.

Therefore, for example, when protein produced by transformant is subjected to the activating treatment, a sulfhydryl compound may be added to a medium for the transformant at any of early stage, middle stage or final stage of the incubation followed by incubating to activate, a sulfhydryl compound may be added to a culture medium after completion of the incubation followed S: by incubating to activate, or a protein is separated from the culture medium and the protein separated therefrom is subjected to a treatment with a sulfhydryl compound to activate.

The present invention will now be further illustrated by way of the following examples although they are just exemplary and do not limit the scope of the present invention.

Example 1.

As a recombinant microorganism, was used Brevibacillus choshinensis HPD31 transformed with plasmid pHT11OEGF prepared by Miyauchi, et al. by using a recombiant human epidermal growth factor gene (Journal of Industrial Microbiology and Biotechnology, volume 21, pages 208-214 (1998)). The medium used for the incubation was prepared by dissolving 40 g of soybean peptone, 5 g of yeast extract, 1.23 g of MgSO 4 .7H 2 0, 0.24 g of MnSO 4 .5H 2 0, 0.01 g of FeSO 4 .7H 2 0, and g of glucose in distilled water to make one liter of the medium.

Each 100 ml of the above-prepared medium was placed in two 500-ml Sakaguchi flasks and sterilized in an autoclave at 121 0 C for minutes. After the sterilization, filter-sterilized erythromycin was added to the medium at 10 mg/liter. Each 1 ml of a seed culture which was incubated overnight in the same medium was inoculated to each sterilized medium and subjected to a shake culture at 33 0 C and 125 rpm. The culture medium was partially sampled from time to time, each of the samples taken was centrifuged and the supernatant was recovered. At the stationary phase in the incubation, i. after 45 hours from the initiation of the incubation, reduced glutathione was added to the medium so that the concentration thereof in the medium could become 1 mM or 5 mM.

Production of active EGF (native-form EGF) was compared with that of the control which was a medium without containing reduced glutathione. The amount of the active EGF produced was determined by means of HPLC. Conditions for the HPLC analysis were that Licrospher 100, RP-18 column (4 x 125 mm) (manufactured by Merck) was used and a concentration gradient of from 25% to 34% of acetonitrile containing 0.1% of trifluoroacetic acid was applied. The flow rate was ml/minute and the detection was carried out at 280 nm. The production of the active EGF was calculated from the area of the peak appeared within an eluting time of the standard hEGF.

The production of the active EGF by incubation is shown in Fig.

1 in terms of the relation between the incubating time (hours; 12 abscissa) and the production of active EGF (grams/liter; ordinate). In order to examine the total amount of EGF produced in each incubated medium, DTT (dithiothreitol) was added to each incubated medium to convert throughly inactive EGF present in each incubated medium into active EGF. Said DTT was added so that the concectration thereof in the medium could become mM. Then, the total amount of active EGF, which is composed of EGF produced in an active form by B. choshinensis HPD31, EGF converted form inactive EGF by reduced glutathione added in the incubation and EGF converted from inactive EGF by 0 DTT above-mentioned (provided that the Control dose not contain was measured by HPLC. The results are shown in Table 1.

Table 1 shows that the addition of reduced glutathione did not effected change in the total amount of EGF (active EGF and inactive EGF produced by B. choshinensis HPD31). Fig. 1 shows that the amount of active EGF obtained increased as the amount of reduced glutathione added increased; said active EGF is the total of and above-mentioned.

Table 1. Changes in Production of Total EGF (g/liter) Concentration of Incubating Time Reduced Glutathione (hours) (mM) 48 56 0 1.4 1.6 1.8 1 1.5 1.7 1.9 1.3 1.7 1.9 Effects of the Invention According to the present invention, inactive protein can be converted into active protein by the very easy treatment, bringing inactive protein into contact with a sulfhydryl compound. Therefore, according to the present invention, protein (often containing inactive protein, thus reducing the yield of active protein and, in some cases, leading to nil) produced by chemical synthesis, biosynthesis or recombinant DNA technology, etc. is contacted with a sulfhydryl compound, thereby inactive protein being converted into active protein and, as a result, the yield of the native-form protein being raised.

For example, in the manufacture of the desired protein by incubation (culturing) of a transformant, inactive protein present in the culture products is converted into active one by a very simple operation that the incubation is carried out by using a medium containing a sulfhydryl compound added to the medium whereupon the desired native-form protein having biological activity can be manufactured in large quantities.

Claims (1)

14- The claims defining the invention are as follows: 1. A method for the manufacture of a biologically active protein, characterized in that a transformant is incubated such that the transformant secretes an exogenous protein into the incubation medium, wherein said exogenous protein, when secreted from said transformant is biologically inactive, and wherein a sulfhydryl compound is added to the medium which when contacted with said biologically inactive protein converts the biologically inactive protein to a biologically active protein, and wherein said biologically active protein is recovered from the medium. 2. The method according to claim 1, wherein the exogenous protein is an epidermal growth factor. 3. The method according to any one of claim 1 or claim 2, wherein the sulfhydryl compound is at least one member selected from the group consisting of reduced glutathione, cysteine, 2-mercaptoethanol and dithiothreitol. 4. The method according to any one of claims 1 to 3, wherein the sulfhydryl compound is added after the logarithmic growth phase in the incubation. The method according to any one of claims 1 to 4, wherein the transformant is prepared by using Brevibacillus choshinensis as the host. 6. A method for the manufacture of a biologically active protein, substantially as described herein with reference to the accompanying drawing. 20 7. A method for the manufacture of a biologically active protein, substantially as described herein with reference to the example. Dated this 9 t h day of September, 2003 S 25 HIGETA SHOYU CO., LTD. By their Patent Attorneys: CALLINAN LAWRIE *o 05/09/03,swl 1040spa.doc,14