DE4329756A1 - Process for the preparation and purification of alpha-interferon - Google Patents

Abstract

The invention relates to a process for preparing recombinant IFN- alpha . Expression in E. coli takes place under control of a phoA promoter. The linkage of the IFN- alpha gene with the STII signal sequence results in secretion of the protein into the periplasmic space and a correctly processed N terminus. The protein is purified by adsorption chromatography on silica gel, hydrophobic interaction chromatography, cation and anion exchange chromatography.

Description

The invention relates to a production method for interferon-α (IFNα) by bacterial Expression and subsequent isolation, an expression vector therefor and a method ren for the purification of IFNα.

Methods for producing IFNα by bacterial expression are known. The usual The method is based on the cytoplasmic expression of the protein in Escherichia coli, at which the expressed IFNα either in insoluble form in so-called inclusion bodies is present in the cell or in the soluble fraction after disrupting the cell wall (Thatcher et Panayotatos, 1986; Goeddel et al., 1980; Dworkin-Rastl et al., 1983). However, cytoplasmic expression has disadvantages. The synthesized Protein is not folded correctly and because of reducing conditions in the cytoplasm it does not contain the required disulfide bridges. The IFNα formed must therefore are oxidized and folded during preparation. This process is inefficient and leads to unwanted by-products (fully or partially reduced forms, oligomers by in Termolecular disulfide bridging, misfolded forms due to the formation of incorrect ones Disulfide bridges), which are difficult to separate. Another problem is that the Nth minimal methionine with which translation begins from the intracellularly synthesized IFNα is only split off incompletely. The resulting N-Met-IFNα can be obtained from the nati ven IFNα practically not be separated.

Another disadvantage of currently used methods is the use of Promoto ren, which are not completely switched off in the non-induced state, by addition of chemicals must be induced, and their expression rate in the induced Zu was not satisfactory, such as B. the trp promoter from Serratia marcescens.

In order to overcome some of the disadvantages mentioned and still the economic E. coli Breitling et al. (Breitling et al., 1989) IFNα1 and an IFNα To express a 1/2 hybrid with a vector that expresses the secretion of the interferon by the Cell membrane in the periplasmic space. They used a promoter Ribosome binding site (RBS) and signal sequence of a bacterial staphylokinase gene (sak42D). 60-80% of the IFNα so produced was in the periplasmic space secreted. However, due to the vector construct, the protein contained additional ones N-terminal amino acids that are not found in the corresponding native IFNα. As gra The disadvantage of this expression system, however, was the fact that the strains transformed from this construct remained genetically unstable; the expressions cassette was deactivated by the spontaneous insertion of an IS1 element. The task, an expression / secretion system in E. coli for the production of human IFNα To deliver, was therefore unsolved in the prior art.  

As an expression / secretion cassette, which in the case of expression of the human Growth factor receptor in E. coli had been a construct from which Promoter of alkaline phosphatase (phoA) and the signal sequence of the heat-stable Entertoxins II (STII) known (Fuh et al., 1990).

Another problem in the production of recombinant IFNα in E. coli is reini delivery of the protein from the bacterial lysate. A number of methods are known here (Thatcher et Panayotatos, 1986; EP-A 203 382). To the native folding of the protein hold, procedures are preferred that do not involve denaturing and precipitation steps get along. Such a method is described in EP-S 396 555. It consists of the steps of immunoaffinity chromatography, reversed-phase chromatography (RPC), Cation exchange chromatography, concentration by ultrafiltration and gel filtration tion chromatography. This method, like other known methods, is based on the ho Selectivity of immunoaffinity chromatography in the first step. It is not a procedure Ren for the production of highly purified IFNα, in particular IFNα2, known without Denaturation / precipitation steps and without immunoaffinity chromatography. At the same time, such a process is desirable for economic and technical reasons worth it. Because of the monoclo necessary for immunoaffinity chromatography Nale antibodies are costly, at the same time, because the lifespan of the antibodies per-coupled matrices is finite, a continuous supply of this antibody necessary.

The object of the invention is to provide a more economical and powerful Process for the production of interferon-α, in particular interferon-α2, by recombinant ante expression in E. coli. The problem had to be solved, an efficient and stable system for the expression / secretion of the protein in the periplasmic space or to establish the culture medium. Furthermore, a method had to be developed that expressed Protective protein without denaturation / precipitation steps and without the need for Can purify immunoaffinity chromatography.

This object was achieved with the present invention. The establishment of a The construction of the stable expression / secretion system for IFNα in E. coli was successful of a vector, the signal sequence (leader sequence) of the heat-stable enterotoxin II (STII) from E. coli linked to the coding sequence for a mature human inter feron-α, preferably interferon-α2, contains. The expression control is preferably carried out by means of the promoter of the alkaline phosphatase from E. coli (phoA). Proved advantageous there is also the integration of the ribosome binding site of the STII gene. Another Surprising progress could be made by providing a cleaning process for Interferon-α, which consists of the steps adsorption chromatography on silica gel, hydrophobic  Interaction chromatography (HIC), cation exchange chromatography and anions exchange chromatography exists.

One aspect of the invention thus relates to a process for the production of IFNα by bak serial expression using transformed bacterial cells that have a Contain expression vector in which the STII signal sequence linked to an IFNα gene and by isolating the expressed IFNα. Another aspect concerns a bacterium Iellen expression vector for the production of IFNα, which is a construct from the Signalse contains sequence of the STII gene and an IFNα gene, and the use of such Vector for the production of IFNα. A third aspect relates to a cleaning process of IFNα by the chromatographic steps adsorption chromatography on silica gel, hydrophobic interaction chromatography, cation and anion exchange chro matography.

A starting point for the construction of the vector can be a replicable in E. coli The plasmid pAT153 (Twigg et al, 1980) is very suitable good for this purpose. A nucleotide sequence that co. For the signal peptide of the STII gene is state of the art (Picken et al., 1983; Lee et al., 1983). The expert is in the Position, through mutations (substitution, deletion, insertion, addition) variants of this Se quenz without changing their basic properties, and especially those Produce nucleotide sequences that are due to the degeneration of the genetic code for encode the same amino acid sequence of the signal peptide (Sambrook et al., 1989, esp. Chapter 15). A whole series of sequences that code for members of the IFNα family ren, is known (Mantei et al., 1980; Streuli et al., 1980; Goeddel et al., 1981); the homo The logic of the genes encoding them is more than 70%. Further variants of these sequences can be found in nature or using methods from the prior art, e.g. B. by mutagenesis, from the known sequences (Sambrook et al., 1989, especially chapter 15). The term "IFNα" in the sense of the invention therefore includes ne ben the known sequences also those variants whose genes by high homolo gie to the known sequences and are marked for biologically active IFNα encode. The sequence which codes for IFNα2c (Dworkin Rastl et al., 1983; Bodo et Fogy, 1985). Use is also particularly preferred of the phoA promoter to control expression and, moreover, advantageously the inte the ribosome binding site of the STII gene. The sequence of the phoA promoter (Chang et al., 1986; Shuttleworth et al., 1981) and the STII ribosome binding site (Picken et al., 1983; Lee et al., 1983) are known; from these sequences too Produce equivalent variants without further ado. Construction of the vector, Transformation of suitable E. coli strains, fermentation and extraction can be done according to known methods take place (Sambrook et al., 1989). For the expression is at for example the E. coli strain W3110 (E. coli K12 wild type f⁻, λ⁻, IN (rrnD-rrnE) 1) is good  suitable. The pre-culture can be done well in LB medium, the main culture under control of sow supply of nutrients and nutrients up to an OD₅₄₆ of 250 to 280. As well suited An extraction method proved to be used, in which acid-activated biomass was diluted Suspended acetic acid using a homogenizer, with polyethyleneimine, preferably in a concentration of 0.25% (w / v) added, to alkaline pH, preferably pH 10, adjusted, stirred and then the bacteria were separated by centrifugation. The cleaning can be carried out according to methods known per se (Thatcher et Panayotatos, 1986; EP-A 203 382). However, a cleaning method with four is particularly advantageous chromatographic steps, namely adsorption chromatography on silica gel, hydrophobic interaction chromatography, cation and anion exchange chromatography graph. The type 953W from the company turned out to be the gel bed of the silica chromatography Grace was well suited as an eluent was a buffer containing 500-1500 mM tetramethylam monium chloride (TMAC), preferably 800 mM TMAC, can advantageously be used. For the hy drophobic interaction chromatography proved to be good for verifying phenyl sepharosis turning gel bed. The sample application was preferably carried out in the presence of 20% Ammonium sulfate, the column was equi with a buffer containing 30% ammonium sulfate been librated. The IFNα was determined using a linear gradient with a final concentration eluted from 30% ethylene glycol. The cation exchange chromatography was very good at a sulfopropyl ion exchange resin. The sample order was placed with pH 3-5, preferably pH 3, the column was equilibrated to pH 5. IFNα could successfully with a linear saline gradient with an addition of 10% Be eluted with ethylene glycol. As a gel bed for anion exchange chromatography DEAE-Sepharose very advantageous to use, application and elution were carried out at pH 5.5- 6.0, preferably at pH 5.8. A linear saline gradient with one was used for elution Addition of 0.1% Tween 20 works well. It is one of the technical possibilities of Expert, without inventive step one or more gel materials to replace equivalent ones, which are based on the same separation principles, and on these Way to perform the inventive method equivalent.

Surprisingly, by linking the STII signal sequence to the IFNα- Gene a stable expression / secretion system to be established, which with the above a sak42D leader / IFNα combination was not successful. He was particularly successful the expression of this sequence was shown to be under the control of the phoA promoter. The inte The ribosome binding site of the STII gene was found to be related in this context as an additional benefit. Expression can be controlled by controlling the phosphate concentration tion in the medium (phosphate deficiency activates the phoA promoter) the; there is no detectable basal expression when inactivated. Additional Chemicals do not need to be added for activation, the expression rate in the activated state is high. The synthesized protein is in high proportions in the peri  plasmatic space secreted. The secreted protein is correctly folded and contains the authentic N-terminus and the correct disulfide bridges. The SDS gel analysis of the Ex Pression in E. coli W3110 showed that 30-50% of the synthesized IFNα processes correctly were, this corresponds to practically the entire proportion of the secreted protein.

Using the extraction method described in Example 3, 29.3 ± 5.9% of the total IFNα2c detectable in the biomass could be extracted. This corresponded to the observed degree of processing of 30-50%. The extract from the biomass contained 4.5 ± 1.8% IFNα2c, based on the total protein. Silica adsorption chromatography resulted in an IFNα2c pool with an average purity of 16.7 ± 4.4%. Phenyl Sepharose chromatography with a yield of 93.2 ± 7.3% gave an IFNα2c with a purity of 71.2 ± 15.5%. The sulfopropyl ion exchange chromatography gave a yield of 70.9 ± 14.8% and a purity of 97.6 ± 4.6%. The final step, DEAE ion exchange chromatography, resulted in 100% pure IFNα2c with a yield of 86.9 ± 9.2% as characterized below. The data from 6 different purifications are summarized in Tables 1 (yields) and 2 (IFNα2c content). Figure 3 shows characteristic chromatograms of each cleaning step.

340 ± 100 mg of purified IFNα1c were obtained from 1 kg of biomass. The yield of the cleaning process is 56.1 ± 22.2%. The overall yield, based on the IFNα2c content of the biomass, is 14.4%. These data are summarized in Table 3. Fig. 4 shows a typical SDS-PAGE of purified IFNα2c, eluted in the last chromatographic step. The 18 kDa band from IFNα2c is the only visible band. Contaminating bands are not observed. Figure 5A shows a typical reversed phase HPLC chromatogram. The purified IFNα2c elutes as a homogeneous peak at 24.8 minutes. When this material was eluted with a flat acetonitrile gradient ( Fig. 5B), 2 contamination peaks were observed on both sides of the main peak. These shoulders, which contain approximately 1.8% of the total IFNα2c content, represent forms that are oxidized at methionine 111 (first shoulder) or acetylated at the N-terminus (second shoulder).

Table 1

Yields of different cleaning steps in percent IFNα2, which were obtained after the respective cleaning step, are shown for 6 different cleaning procedures (p1-p6) from 6 different biomasses. The last two columns contain the mean (M) and the standard deviation (sd)

Table 2

IFNα2 content of various cleaning steps. The data are as a percentage of IFNα2 content based on total protein content; obtained in this cleaning step, as shown in Table 1

Table 3 Total yields of the cleaning process. The IFNα2c content of the biomass is shown as g IFNα2 / kg biomass. Processing and extraction are expressed as a percentage of the total IFNα2 content. The purification yield is shown as a percentage of IFNα2c relative to the IFNα2 content of the extract. The total yield is expressed in mg IFNα2 obtained per kg of biomass and as a percentage of purified IFNα2c based on the IFNα2c content of the extract

Illustrations

Figure 1A) Gene map of pCF2. The EcoRI-BamHI fragment from pAT153 was replaced by the expression cassette for IFN-ω1.
B) Sequence of the EcoRI (destroyed) BamHI part which contains the phoA promoter, STII-Lea of the + IFNω1 gene.

Fig. 2A) Gene map of the plasmid pDH13. The SspI-PstI fragment from pAT153 was replaced by the IFNα2c expression cassette (EcoRI-PstI fragment from 2B)). The β-lactamase gene has been destroyed.
B) Nucleotide sequence of the EcoRI-HindIII insert from pDH13.

Fig. 3 Chromatographic purification of IFN-α2c, extracted from biomass.
A) Adsorption chromatography on silica gel. The arrow shows elution with 800 mM tetramethylammonium chloride.
B) Hydrophobic interaction chromatography on phenyl sepharose. Elution was carried out with a linear gradient from 0 to 100% solvent B as indicated (----).
C) Sulfopropyl cation exchange chromatography. Elution was carried out with a gradient from 0 to 100% solvent B as indicated (----).
D) Anion exchange chromatography on DEAE-Sepharose. Elution was carried out with a gradient from 0 to 100% solvent B as indicated (----).
The bars under the main peaks in each chromatogram indicate the IFNα2-containing pools that were collected and used for the following steps.

Fig. 4 SDS-PAGE of purified IFNα2c, stained with Coomassie Blue. The numbers on the left indicate the molecular weights of the standard proteins.
Lane 1: IFNα2c standard
Lane 2: 3 µg IFNα2c
Lane 3: 6 µg IFNα2c
Lane M: molecular weight standard.

Fig. 5 Characterization of purified IFNα2c by reversed phase HPLC.
A) Elution of IFNα2c with a linear gradient of 20-68% solvent B in 24 minutes.
B) Elution of IFNα2c with a linear gradient of 45-53% solvent B in 30 minutes.

Examples Example 1: Production of the expression vector pDH13 and thus transformed Cells General methods

Restriction digestion of DNA with restriction endonucleases, replenishment reactions, Phenolex Traction and precipitation of DNA, agarose gel electrophoresis and elution from DNA Agarose gels, ligation of DNA molecules, transformation of bacteria and plasmi Disolation from bacteria are standard procedures and were carried out as by Sam brook et al. (1989).

Plasmids

pCF2 pCF2 was made from plasmid pAT153 (Twigg et al, 1980). It contains the E. coli alkaline phosphatase promoter (phoA, Chang et al., 1986; Schuttleworth et al., 1986), the coding region of the STII leader peptide (Picken et al., 1983; Lee et al., 1983 ) and the gene for human IFNα1 (Hauptmann et al., 1985). Fig. 1 shows the gene map of pCF2 and the sequence of the relevant section.

pER21 / 1 pER 21/1 is a bacterial expression vector for IFNα2c (Dworkin-Rastl et al., 1983)
Oligonucleotides (5 ′ → 3 ′):

Production of the expression cassette from the phoA promoter, IFNα2c sequence and STII-Lea sequence in a two-step PCR

pER21 / 1 DNA was linearized with HindIII, pCF2 DNA with PvuI. The following ver The method used is described as SOE-PCR ("splicing by overlap extension", Ho et al., 1989).

PCR 1a (amplification of the IFN-α2c gene): 100 ng linearized pER21 / 1 DNA, 25 pmol EBI-2797 and 25 pmol EBI-2798 were in 50 ul buffer, the 50 mM KCl, 10 mM Tris HCl, pH 8.3, 1.5 mM MgCl₂, 0.01% gelatin, 0.2 mM ATP, 0.2 mM dGTP, 0.2 mM dCTP, 0.2 mM dTTP and 1.25 units Taq polyinerase contained in a Perkin Elmer Cetus Thermocycler TC-1 subjected to thermal cycles. After 3 min incubation at 94 ° C 10 step cycles (step 1: 40 sec at 94 ° C, step 2: 30 sec at 55 ° C, step 3: 90 sec at 72 ° C).

PCR 1b (amplification of phoA promoter plus STII leader sequence): 100 ng linearized pCF2-DNA, 25 pmol EBI-2787 and 25 pmol EBI 2799 were in the same buffer and un Subject to the same conditions as described under PCR 1a thermal cycles.

The resulting DNA fragments from PCR 1a (540bp) and PCR 1b (374 bp) were gel purified (1.2% low gelling type agarose in TBE buffer, 1 × TBE: 10.8 g Tris / l, 5.5 g boron acid / l, 0.93 g EDTA / l). The piece of agarose, which contained the respective DNA fragment, was cut out and the agarose melted by adding 100 .mu.l H₂O and on 70 ° C was heated.

PCR 2: 5 ul of each agarose / DNA solution were combined and in 100 ul solution each containing 50 pmol of EBI-2787 and EBI-2797, subjected to thermal cycling. Of the Buffer was the same as described under PCR 1a. The thermal cycle device was like this programmed that 20 step cycles at a delay time of 5 min at 94 ° C (step 1: 40 sec at 94 ° C, stage 2: 30 sec at 55 ° C, stage 3: 5 min at 72 ° C; Level 3 was awarded to everyone new cycle extended by 5 seconds). After amplification the DNA was purified by phenol / chloroform extraction and ethanol precipitation. The PCR product was dissolved and with HindIII and EcoRI in the corresponding puf cut away.

Cloning the PCR product (pDH9)

Bluescribe M13⁺ (Stratagene, San Diego, CA, USA) was doubled with HindIII and EcoRI cut and the large fragment was gel cleaned with a 1.2% agarose gel. 10th ng Bluescribe M13⁺ DNA and 50 ng PCR product cut with EcoRI / HindIII were in 10 ul solution containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 20 mM dithio  threitol, 1 mM ATP, 50 µg / ml bovine serum albumin (BSA) and 2 units of T4 DNA ligase (NEN), ligated for 1 hour at 0 ° C and 3 hours at room temperature. 8 µl of this Solution for the transformation of competent E. coli cells from strain JM 101 (E. coli K12, SupE, thi, Δ (lac · proAB), [F ′, traD36, proAB, lacIZΔM15]) was used.

A clone was selected, the DNA isolated and the expression cassette sequenced. The sequence corresponded exactly to the theoretically expected sequence ( FIG. 2). The plasmid was named pDH9.

Construction of the expression plasmid pDH13

pAT153 was double cut with SspI and PstI and the large fragment became iso liert. pDH9 was cut with Eco RI and the ends filled in using the Klenow fragments of DNA polymerase I and the 4 dNTPs. After phenol extraction and Precipitation of the linear pDH9 DNA, this DNA was cut with Pst I and the fragment, which contained the phoA promoter, the STII leader sequence and the IFNα2c gene from one 1% agarose gel isolated.

10 ng pAT153 × SsoI × PstI and 30 ng of the fragment containing the expression cassette were ligated in 10 μl solution for 5 hours at room temperature. 5 µl of this approach was used to transform competent HB101 E. coli bacteria. The selection of the transformed bacteria was carried out on LB agar plates (10 g tryptone / l, 5 g yeast extract / l, 5 g NaCl / l, 15 g Bacto agar / l): which contained 10 μg / ml tet racycline. A gene map of pDH13 and the sequence of the relevant region is shown in Fig. 2.

Plasmid DNA from various colonies thus obtained was isolated and by restriction analysis checked for correct composition. A plasmid was selected and as designated pDH13. The plasmid pDH13 was used to transform E. coli W3110 (E. coli K12 wild type, f⁻, λ⁻, IN (rrnD-rnnE) 1) was used.

Example 2: fermentation Preculture

700 ml autoclaved LB medium (10 g bacto-trypton / l, 5 g bacto-yeast extract / l, 10 g NaCl / l, pH 7.0), which contained 5 mg / l tetracycline, were prepared in a 2 l glass vessel Inoculated stock culture so that an OD₅₄₆ of 0.01 was obtained. The culture turned 10 Hours at 37 ° C with vigorous stirring (800 rpm) and aeration (5 fermenter volumes per minute [vvm]) incubated.  

Main culture Medium composition

in the fermenter:

1.21 g / l (NH₄) ₂HPO₄
3.96 g / l (NH₄) ₂SO₄
6.53 g / l K₂HPO₄
1.23 g / l MgSO₄ × 7H₂O
0.32 g / l NaCl
0.25 g / l NH₄Cl
1.0 g / l Na₃ citrate × 2H₂O
1.0 ml / l trace element concentrate
12.5 g / l glucose
20 mg / l thiamine HCl
50 mg / l L-tryptophan
100 mg / l L-leucine
50 mg / l L-methionine
5 mg / l tetracycline

Trace element concentrate:
(Quantities per 100 ml)

3.35 g FeCl₃ × 6H₂O
1.09 g ZnSO₄ × 7H₂O
0.267 g CoCl₂ × 6H₂O
0.267 g Na₂MoO₄ × 2H₂O
0.221 g CuSO₄ × 5H₂O
0.333 g H₃BO₃
1.37 g MnSO₄ × H₂O
10 ml HCl conc.
H₂O ad 100 ml

Feeding during fermentation:
(Quantities based on fermenter volume)

350 g / l glucose
3.70 g / l MgSO₄ × 7H₂O
175 mg / l thiamine HCl
0.50 g / l L-tryptophan
4.0 g / l L-leucine
2.0 g / l L-methionine
Antifoam dosage during fermentation:
(based on fermenter volume)

1.0 ml / l UCON LB625

Salts ((NH₄) ₂PO₄, (NH₄) ₂SO₄, K₂HPO₄, NaCl, NH₄Cl and Na citrate) were combined in one Sterilized fermenter. Trace elements, MgSO₄, glucose, thiamine, L-tretophan, L-leucine, L-methionine and tetracycline were added aseptically after cooling so that a Initial volume of 7 liters was obtained. 600 ml of the pre-culture were automatically in  inoculated the fermenter. The fermentation conditions were: stirring at 1000 rpm, Aeration of 1 vvm, 0.3 bar overpressure, a temperature of 37.0 ± 0.1 ° C, the pH was kept at 6.7 ± 0.1 with NH₃ and H₂SO₄. The concentration of dissolved oxygen was increased by ventilation with oxygen-enriched air as required above 15% Air saturation (at 0.3 bar back pressure overpressure. After consumption the initially existing glucose, a feeding procedure was started by the sow concentration was triggered automatically and glucose, thiamine, MgSO₄, L- Contained tryptophan, L-leucine and L-methionine. The feeding speed started with 2.5 g / l * h glucose and was continuously increased to 5.0 g / l * h within 24 hours and then kept constant until the end of the fermentation process.

The fermentation was stopped after a total of 350 g / l of glucose had been added had been given. At that time, a typical optical density was 250 to 280 reached at 546 nm.

To inactivate the biomass, the batch was cooled to about 10 ° C. and simultaneously set the pH to 2.0 with H₂SO₄. The biomass was removed by centrifugation separated and stored frozen at -70 ° C.

Example 3: Extraction

Acid-activated biomass (about 0.5 kg) was egg in 500 ml of 1% acetic acid nes Polytron homogenizer suspended and stirred at 0 ° C for 1 hour. Polyethyleneimine (50% stock solution, Serva, Heidelberg) was up to a final concentration of 0.25% (w / v) added. The suspension was adjusted to a pH of 10.0 with 5 N NaOH and stirred for a further 2 hours at 0 ° C. After adjusting the pH to 7.5 with 5 N HCl the bacteria by centrifugation at 17000 × g (Beckmann J2-21 centrifuge) severed. The average extraction yield was 29.3 ± 5.9% of the total stop at IFNα2c.  

Example 4: Chromatographic purification Adsorption chromatography on silica gel

The IFNα-containing supernatant after separation of the bacterial pellet in Example 3 was loaded onto a silica gel column (Grace, silica type 953W; 35 mg protein / ml column material, flow rate 25 ml / mm), which was treated with 20 mM Tris-HCl, pH 7.5, had been equilibrated. The column was washed with 30 column volumes of start buffer, followed by a washing step with 20 mM Tris-HCl, 100 mM tetramethylammonium chloride (TMAC), pH 7.5. IFNα2c could be eluted by increasing the TMAC concentration to 800 mM TMAC ( Fig. 3A).

Hydrophobic interaction chromatography

The material that eluted from the silica gel column was removed by adding solid (NH₄) ₂SO₄ adjusted to an ammonium sulfate concentration of 20% (w / u) and to one Phenyl Sepharose column (Phenyl Toyopearl, 650S, Tosohaas) loaded with 20 mM Tris HCl, 30% ammonium sulfate had been equilibrated. IFNα2c was linear Gradients from 100% loading conditions to 100% 20mM Tris-HCl, 30% ethylene glycol, pH 7.5, eluted at a flow rate of 15 ml / min. The purity of the IFNα pool was 71 ± 15%.

Cation exchange chromatography

The eluate of the hydrophobic interaction chromatography was revealed by extensive dialysis 20 mM Na succinate, pH 5.0. The final pH was adjusted to 3.0 with HCl before placing the sample on a sulfopropyl ion exchange resin (Toyopearl TSK SP 5PW, Tosohaas), equilibrated with 20 mM Na succinate, pH 5.0. IFNα2c was with a linear gradient from 100% loading conditions to 100% 20 mM Na succinate, 500 mM NaCl, 10% ethylene glycol, pH 5.5 (solvent B) with a flow rate of 6 ml / min eluted from the column. The IFNα2c eluted from this column was routine a purity greater than 95%.

Anion exchange chromatography

The IFNα pool was dialyzed against 10 mM bisTris, pH 5.8 and tested for a DEAE-Se pharose (DEAE-Sepharose FastFlow, Pharmacia) loaded with the same buffer was equilibrated. IFNα2c was eluted with a linear gradient to 10 mM  bisTris, 500 mM NaCl, 0.1% Tween 20, pH 5.8 (solvent B), flow rate 5 ml / min.

Example 5: Analysis of IFNα2c preparations Reversed phase HPLC

IFNα2c was intact with a BakerBond -WP- C18 column [250 × 4.5 mm, particles size 5 µm] analyzed at 30 ° C. One was used for the separation of tryptic peptides Merck Supersphere 120-4 C-18 column [125 × 4.5 mm, particle size 4 µm] at 37 ° C ver turns. The samples were made using solvent A, 0.1% trifluores acetic acid in water, and B, 0.1% trifluoroacetic acid in acetonitrile and with the gradients chromatographed as described in the relevant figure legend.

SDS polyacrylamide gel electrophoresis

IFNα2c samples were on 16% SDS polyacrylamide gels under standard conditions analyzed. Samples were reduced with dithiotreitol before electrophoresis. Protein bands were visualized with Coomassie Blue staining.

Quantification of IFNα2c by ELISA

The IFNα2c content of various samples that were obtained during the cleaning was determined with a sandwich ELISA with the monoclonal antibodies OMG-2 and MG-7 (Adolf et al, 1990).

literature

Adolf, GR, Virology 175, 410-417 (1990)
Bodo, G. & Maurer-Fogy, I., in: The Biology of the Interferon System 1985 (ed .: Ste wart. WE, II & Schellekens, H.), pp. 59-64, Elsevier Scientific Publishing Co., Amsterdam (1985)
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Chang CN, Kuang W.-J. and EY Chen, Gene 44, 121-125 (1986)
Dworkin-Rastl E., Swetly P., Dworkin MB, Gene 21, 237-248 (1983)
Fuh G., Mulkerrin MG, Bass S., McFarland M., Brochier M., Bourell JH, Light DR, Wells JA, J. Biol. Chem. 265, 3111-3115 (1990)
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Claims (25)

1. A method for producing interferon-α by expression in E. coli, characterized in that

• a) Interferon-α is expressed in cells which contain a vector in which the signal sequence of the gene for the heat-stable enterotoxin II (STII) from E. coli is linked to a sequence which codes for mature human interferon-α
• b) the expressed interferon-α is isolated.

2. The method according to claim 1, characterized in that the vector additionally a Contains promoter for alkaline phosphatase (phoA) from E. coli. 3. The method according to claims 1 to 2, characterized in that the vector additionally contains the sequence for the ribosome binding site of the STII gene. 4. The method according to claim 1 to 3, characterized in that the isolation of the interferon in the steps

• a) Adsorption chromatography on silica gel
• b) Hydrophobic interaction chromatography
• c) Cation exchange chromatography
• d) anion exchange chromatography

contains. 5. The method according to claim 4, characterized in that the hydrophobic interac tion chromatography is carried out on a phenylsepharose column. 6. The method according to claim 4, characterized in that the cation exchange chromatography is carried out on a sulfopropyl ion exchanger. 7. The method according to claim 4, characterized in that the anion exchange chromatography is carried out on a DEAE-Sepharose.   8. The method according to claims 1 to 7, characterized in that the interferon α is interferon-α2. 9. The method according to claim 8, characterized in that the interferon-α2 the amino acid sequence contains. 10. Process for the purification of interferon-α, characterized in that it has the steps

• a) Adsorption chromatography on silica gel
• b) Hydrophobic interaction chromatography
• c) Cation exchange chromatography
• d) anion exchange chromatography

contains. 11. The method according to claim 10, characterized in that the hydrophobic interac tion chromatography is carried out on a phenylsepharose column. 12. The method according to claim 10, characterized in that the cation exchange chromatography is carried out on a sulfopropyl ion exchanger.   13. The method according to claim 10, characterized in that the anion exchange chromatography is carried out on a DEAE-Sepharose. 14. The method according to claims 10 to 13, characterized in that the interferon-α was expressed bacterially. 15. The method according to claims 10 to 14, characterized in that the inter feron-α is interferon-α2. 16. The method according to claim 15, characterized in that the interferon-α2 the amino acid sequence contains. 17. Vector for the expression of interferon-α in E. coli, characterized in that it contains the signal sequence of the STII gene linked to a sequence which is for mature human interferon-α encoded. 18. Vector according to claim 17, characterized in that it additionally has a phoA Contains promoter. 19. Vector according to claims 17 to 18, characterized in that it additionally the Contains ribosome binding site of the STII gene.   20. Vector according to claims 17 to 19, characterized in that the interferon α is interferon-α2. 21. Vector according to claims 17 to 19, characterized in that it has the nucleotide sequence or contains a sequence which is more than 70% homologous to this sequence and which codes for interferon-α. 22. Vector according to claim 21, characterized in that it contains the nucleotide sequence contains. 23. Use of the vector according to one of claims 17 to 22 for the production of Interferon-α.