KR20000065580A - Process for Preparing Amino-terminal Methionine Free Recombinant Interferon-alpha - Google Patents

Abstract

PURPOSE: Provided is a preparation method of recombinant interferon-alpha of which methionine at amino end is eliminated. Thereby the recombinant interferon-alpha is prepared easily which is suitable for a therapeutic recombinant protein for administration and which is safe in vivo. CONSTITUTION: A method for preparation of recombinant interferon-alpha of which methionine at amino end is prepared by: treating aminopeptidase originated from Aeromonas proteolytica into recombinant interferon alpha to remove methionine at amino end and reacting at 20-30°C for 12-20 hours; isolating and purifying using cation exchange chromatography; and expressing the recombinant interferon alpha in the recombinant E.coli δpMAIFN alpha, KCCM 10053.

Description

Process for Preparing Amino-terminal Methionine Free Recombinant Interferon-alpha}

The present invention relates to a method for producing a recombinant interferon-α from which the methionine present at the amino terminus is removed from the transformed recombinant E. coli. More specifically, the present invention comprises the step of treating recombinant interferon-α with an aminopeptidase to remove amino-terminal methionine and purifying using cation-exchange chromatography. The present invention relates to a method for producing a recombinant interferon-α from which methionine present at an amino terminus is removed from recombinant E. coli.

With the development of genetic engineering, methods for producing necessary proteins using recombinant microorganisms have been rapidly developed. However, in general, a protein product expressed by genetic recombination technology produces a non-natural recombinant protein having methionine (N-met) in addition to the amino acid sequence designated by the gene of the protein product at its amino terminus. No exception. Recombinant proteins do not have the same structure as proteins in the human body, so when the recombinant recombinant protein for body administration is produced by E. coli, N-met causes an unexpected immune response from the host or the protein itself It was unstable and could not fully function. For example, human growth hormone composed of 191 amino acids secreted by the human pituitary gland and whose amino terminus starts with phenylalanine-proline-threonine (Phe-Pro-Thr) has been produced by genetic recombination and used in the treatment of dwarfism. However, in the recombinant human growth hormone, in addition to 191 amino acids, methionine has not been removed at the amino terminus in many cases, and it has been reported that unexpected antibody formation is caused by the human growth hormone added with methionine.

Therefore, various methods have been developed to solve the problem of such recombinant proteins, for example, a method for producing and cleaving a recombinant fusion protein to remove methionine located at the amino terminus (see WO 89/12678, EP 20209). , EP321940), or a method (see EP 008832, USP 4,755,464) for cutting methionine in the process of secreting the recombinant protein out of the host cell has been proposed. However, these methods expose the problem that additional manipulation such as re-engineering of expression vectors and transformation of host cells is necessary and conditions for optimization of fermentation should be set.

Meanwhile, a method of preparing a native protein using a method of aminopeptidase, an enzyme that selectively cleaves only methionine located at the amino terminus of a recombinant protein, has been proposed (see WO 86/01229 and WO 86). / 204527, EP6296695, USP 5,569,598). According to this method, however, the recombinant protein could be converted to a naturally occurring protein relatively simply. However, in order for the aminopeptidase to act on the recombinant protein and convert to a native protein, only the amino terminal methionine was removed. The cleavage reaction must be stopped, and a small amount must be able to convert the recombinant protein into a natural protein, and the methionine removal activity of the aminopeptidase is greatly affected by the type of adjacent amino acid following methionine. Was doing. Therefore, there is a constant need to develop a method for efficiently removing and purifying amino-terminal methionine according to the structural and biochemical properties of each recombinant protein.

Accordingly, the present inventors have efficiently removed the amino terminal methionine of recombinant interferon-α expressed from E. coli, thereby producing a recombinant interferon-α in which the amino terminal methionine is removed more reliably and clinically safer in human body as a recombinant protein. As a result of intensive efforts, the recombinant interferon-α can selectively remove only amino-terminal methionine due to its structural specificity, and it is also a natural type of interferon-α in which amino-terminal methionine is removed through cation exchange chromatography. It was confirmed that can be easily purified, and to complete the present invention.

After all, the main object of the present invention is to provide a method for producing a recombinant interferon-α from which the methionine present at the amino terminus is removed from the transformed E. coli.

1 is a gel photograph by iso-electrofocusing (IEF) of partially purified interferon-α.

Figure 2 is a gel photograph by IEF of a sample separated by cation exchange chromatography after aminopeptidase treatment of partially purified interferon-α.

Figure 3 is a chromatogram showing the amino acid sequence analysis of the amino terminal of the partially purified interferon-α.

4 is a chromatogram showing the amino acid sequence analysis of the amino terminal of the sample separated by cation exchange chromatography after aminopeptidase treatment to partially purified interferon-α.

In the present invention, a method for preparing recombinant interferon-α from which amino terminal methionine is removed is treated with aminopeptidase to partially purified recombinant interferon-α, and purified methionine-removed recombinant interferon-α using cation exchange chromatography. It includes a process to make.

In the present invention, recombinant interferon-α is prepared in which amino terminal methionine is removed from partially purified interferon. In the following example, the interferon-α (refer to Korean Patent No. 134856) expressed in the form of inclusion body from the transformed recombinant E. coli was dissolved in 8M urea and then refolded, and Cu2 + Although the example employs a partially purified interferon-α by carrying out metal-chelate affinity chromatography with chelate sepharose and ion exchange chromatography with SP sepharose, the amino terminal methionine of the present invention The range of partially purified interferon-α that can be used in the preparation of this removed recombinant interferon-α is not limited to the examples described below, and includes interferon-α partially purified from various recombinant protein expressors.

After adjusting the pH of the partially purified interferon-α sample, ZnCl2 was added and the aminopeptidase was added at 0.2 to 10 units per mg of the partially purified protein, and then at 20 to 30 ° C, most preferably at 25 ° C. For 12 to 20 hours, more preferably 14 to 18 hours, most preferably 16 hours to remove the amino terminal methionine. After the pH of the aminopeptidase-treated sample obtained in the above process was lowered to 4.0, the sample diluted twice with the buffer solution was filtered through a 0.45 μm filter, and then the cation was prepared using an ion exchange resin having strong ion exchange properties. Exchange chromatography is performed. Subsequently, eluted stepwise using a buffer solution of 50 to 200 mM NaCl, and the fraction eluted from the amino-terminal methionine was confirmed by isoelectrofocusing (IEF). Afterwards, interferon-α is obtained.

The amino terminal methionine (N-met) of interferon-α can be specifically removed by the aminopeptidase due to the disulfide bond structure of interferon-α, and interferon-α is divided into two disulfide bonds; cys1-cys98, cys29-cys138). Those with both disulfide bonds are called fast monomers, those with only one (cys29-cys138) are called slow monomers, and both forms are identical in biological activity and most Interferon-α exists as a fast monomer. Since the first cysteine of the fast monomer forms an internal bridge with the 98th cysteine by disulfide bond, the enzymatic reaction is stopped after N-met is removed by aminopeptidase treatment. It provides a principle that only N-met can be specifically removed by the aminopeptidase used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by the examples according to the gist of the present invention.

Example 1 Obtaining Partially Purified Interferon-α

In order to obtain partially purified interferon-α, recombinant E. coli (E. coli ΔpMAIFNα, KCCM) transformed with a plasmid in which a human interferon-α gene was inserted into an inducible expression vector using L-arabinose operon of Salmonella strains. 10053) was dissolved in 8M urea solution of interferon-α (refer to Korean Patent No. 134856) expressed in the form of an inclusion body, and then gradually diluted to perform the refolding process. Activated interferon-α was adsorbed onto Cu 2+ -chelate Sepharose (Cu2 + -chelate Sepharose, Pharmacia Biotech, Sweden) equilibrated with adsorption solution (20 mM phosphate buffer (pH 7.2) containing 1 M NaCl), and then 5 to Eluted with 20 mM imidazole. The eluted sample was concentrated through ultrafiltration, and then attached to SP Sepharose (SP Sepharose, Pharmacia Biotech, Sweden) to elute with 50 to 250 mM NaCl to obtain partially purified interferon-α. In the partially purified interferon-α samples, amino-terminal amino acids were sequenced using 492 Protein Sequencer (Perkin Elmer, U.S.A.). As a result, at least 30% of the total interferon-α had N-met.

Example 2: Confirmation of Interferon-α Separation Using Isoelectric Point Fractionation Electrophoresis

The sample and the pI standard sample partially purified according to Example 1 were placed in each well of an IEF gel (NOVEX, USA) having a pI range of 3 to 9, and then 1 hour at 100V, 1 hour at 200V, and 30 minutes at 500V. It was developed and subjected to IEF gel electrophoresis. Cathode buffer and anode buffer were added to the cathode and anode, respectively, and developed in the same manner as the NOVEX guidelines (pp.19-20, NOVEX pre-cast gel instruction), and caps (3- [cyclohexylamino]-was developed. The PVDF membrane was electrophoreted to PVDF membrane (Biorad, USA) via 1-propane-sulphonic acid) buffer solution (composition: 10 mM caps (pH 11), 10% methanol), and the protein was transferred to the PVDF membrane. Staining with Coomassie Brilliant Blue confirmed the respective band positions (see FIG. 1). In Figure 1, lane 1 is partially purified interferon-α; And lane 2 represent pI standard samples, respectively. At this time, each band electrophoresis was cut out by the PVDF membrane, and the amino acid sequence of the amino terminal was analyzed using a 492 Protein Sequencer (Perkin Elmer, U.S.A.) (see Table 1). As shown in Table 1 and FIG. 1, interferon-α (met-IFN) having methionine at its amino terminal is present at a high ratio in the upper band near pI 6.2 and in the middle and lower bands, interferon- completely removed methionine. only α (IFN) was present. This suggests that met-IFN and IFN can be separated by ion exchange chromatography.

Amino acid sequencing result of amino terminal of partially purified interferon-α division Amino Acid Detection Sequence One 2 3 4 Upper band Met Asp Leu Pro Middle band X Asp Leu Pro Bottom band X Asp Leu Pro

X: no amino acids detected

Example 3: Aminopeptidase Treatment

After adjusting the pH of the partially purified interferon-α sample to 8.0, ZnCl2 was added to 0.01 to 0.1 mM, and aminopeptidase (Sigma, USA) derived from Aeromonas proteolytica was added. 0.2 to 10 units per mg of partially purified interferon-α protein was reacted at 25 ° C. for 16 hours. Subsequently, IEF gel electrophoresis was performed in the same manner as in Example 2 to confirm the removal of the amino terminal methionine (see FIG. 2). Lane 1 in Figure 2 is the pI standard sample; Lane 2 is a partially purified interferon-α sample; Lane 3 is an interferon-α sample separated by Resource S; And lane 4 is a sample obtained by treating aminopeptidase with partially purified interferon-α. As shown in Figure 2, the aminopeptidase treatment by the above method, it was confirmed that the met-IFN band is converted to the IFN band on the IEF.

Example 4: Cation Exchange Chromatography

As in Example 3, the pH of the aminopeptidase-treated sample was lowered to 4.0, and then diluted twice with 20 mM sodium phosphate buffer solution (hereinafter referred to as 'buffer 1') having a pH of 4.3 to 5.5, The diluted sample was filtered through a 0.45 μm filter and passed through a Resource S (Pharmacia Biotech, Sweden) column equilibrated with Buffer 1. The sample attached to Resource S was eluted step by step using Buffer 1 to which 50 to 200 mM NaCl was added, and the fraction of met-IFN was completely removed using IEF as in Example 2 using the sample eluted step by step. It was confirmed (see Figure 2, lane 3), and finally compared to before and after the process by amino acid sequencing of the amino terminal, it was confirmed that the N-met was completely removed (see Figure 3, Figure 4). As shown in the amino terminal sequence analysis of the amino terminal of Figure 3, it was confirmed that a significant portion of the partially purified interferon-α has a methionine at the amino terminal, as shown in Figure 4 separated by Resource S after aminopeptidase treatment No amino acids were detected in the first amino terminal sequence of one interferon-α. The fact that no amino acids were detected when sequencing through the Edman degradation process described above meant that methionine was removed through this process, resulting in the first amino terminal sequence of interferon-α (IFN) becoming cysteine. This first cysteine is connected to cysteine 98 by forming an internal bridge through disulfide bonds. Aspartic acid was detected as the second amino acid detected, and the result was consistent with that previously reported (HS Park, Korean Biochem. J., Vol. 20, No. 4, pp. 336-344). , 1987).

As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described and demonstrated in detail above, the present invention provides a method for preparing a recombinant interferon-α, wherein the amino terminal methionine is removed from the transformed recombinant E. coli. According to the present invention, the recombinant interferon-α from which the amino terminal methionine is removed can be produced more reliably and clinically and more securely suitable for a recombinant protein for therapeutic purposes intended for in vivo administration.

Claims (6)

Preparation of the amino-terminal methionine-free recombinant interferon-α, which comprises a step of treating and reacting the recombinant interferon-α with an aminopeptidase and then purifying the recombinant interferon-α from which methionine has been removed by cation exchange chromatography. Way. The method of claim 1, Recombinant interferon-α is characterized in that it is expressed from recombinant E. coli (E. coli ΔpMAIFNα, KCCM 10053) A method for preparing recombinant interferon-α, wherein amino terminal methionine is removed. The method of claim 1, Aminopeptidase, characterized in that derived from Aeromonas proteolytica (Aeromonas proteolytica) A method for preparing recombinant interferon-α, wherein amino terminal methionine is removed. The method of claim 1, The reaction is characterized in that carried out for 20 to 30 ℃, 12 to 20 hours A method for preparing recombinant interferon-α, wherein amino terminal methionine is removed. The method of claim 1, Resin of cation exchange chromatography is Resource S, characterized in that A method for preparing recombinant interferon-α, wherein amino terminal methionine is removed. The method of claim 5, Cation exchange chromatography is eluted using a 20 mM sodium phosphate buffer solution with a pH of 4.3 to 5.5 to which 50 to 200 mM NaCl is added. A method for preparing recombinant interferon-α, wherein amino terminal methionine is removed.