Method for producing a recombinant protein of interest by using the Npro technology

Abstract

Disclosed is a method for producing a recombinant protein of interest, the method being characterized in by the following steps: (a) providing a fusion protein comprising an N.sup.pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilizing the inclusion bodies, (c) allowing the fusion protein to be cleaved by the N.sup.pro autoprotease moiety under chaotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein and wherein the recombinant protein of interest is not yet renatured or simultaneously renatured, and (d) recovering the protein of interest, optionally including a renaturing step for the protein of interest.

Claims

1. Method for producing a recombinant protein of interest, which comprises: (a) providing a fusion protein comprising an N.sup.pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the inclusion bodies, (c) allowing the fusion protein to be cleaved by the N.sup.pro autoprotease moiety under chaotropic conditions, wherein the chaotropic conditions correspond to a urea concentration of 2 to 5 M urea, wherein the recombinant protein of interest is cleaved from the fusion protein and wherein the recombinant protein of interest is not yet renatured or simultaneously renatured, and (d) recovering the protein of interest, optionally including a renaturing step for the protein of interest, wherein the N.sup.pro autoprotease moiety consists of the sequence set forth by SEQ ID NO: 1.

2. The method according to claim 1, wherein the inclusion bodies were generated in a recombinant production system.

3. The method according to claim 1, wherein the conditions in step (b) correspond to a urea concentration of more than 5 M.

4. The method according to claim 1, wherein the chaotropic conditions in step (c) correspond to a urea concentration of from 2 to 4 M.

5. The method according to claim 1, wherein the N.sup.pro autoprotease moiety has a 24 h cleavage rate at 2.5 M urea of at least 20%.

6. The method according to claim 1, wherein the protein of interest is a protein for therapeutic use in humans.

7. The method according to claim 1, wherein step (b) and/or step (c) is performed at a pH of 5 to 11.

8. The method according to claim 1, wherein step (b) is performed under basic pH conditions.

9. The method according to claim 1, wherein the renaturing step for the protein of interest is carried out after recovery of the protein of interest.

10. The method according to claim 1, wherein the fusion protein comprises additional moieties.

11. The method according to claim 1, wherein the fusion protein is at least partially purified between step (b) and step (c).

12. The method according to claim 1, wherein the fusion protein is at least partially purified between step (b) and step (c) by affinity purification.

13. The method according to claim 1, wherein in step (b) is performed in the presence of guanidinium hydrochloride at a concentration of more than 2.5 M.

14. The method according to claim 1, wherein in step (c) is performed in the presence of guanidinium hydrochloride at a concentration of from 0.7 to 2.5 M.

15. The method according to claim 2, wherein the recombinant production system is a prokaryotic host cell.

16. The method according to claim 2, wherein the recombinant production system is E. coli host cells.

17. The method according to claim 3, wherein the urea concentration in step (b) is more that 6 M.

18. The method according to claim 17, wherein the urea concentration in step (b) is more that 7.5 M.

19. The method according to claim 5, wherein the N.sup.pro autoprotease moiety has a 24 h cleavage rate at 2.5 M urea of at least 30%.

20. The method according to claim 19, wherein the N.sup.pro autoprotease moiety has a 24 h cleavage rate at 2.5 M urea of at least 40%.

21. The method according to claim 6, wherein the protein of interest is a human recombinant protein or a vaccination antigen.

22. The method according to claim 7, wherein step (b) and/or step (c) is performed at a pH of 6 to 9.5.

23. The method according to claim 22, wherein step (b) and/or step (c) is performed at a pH of 6.5 to 8.5.

24. The method according to claim 11, wherein step (b) is performed in the presence of more than 5 mM NaOH or KOH.

25. The method according to claim 24, wherein step (b) is performed in the presence of more than 25 mM NaOH or KOH.

26. The method according to claim 25, wherein step (b) is performed in the presence of more than 100 mM NaOH or KOH.

27. The method according to claim 1, wherein the renaturing step for the protein of interest is carried out after recovery of the protein of interest.

28. The method according to claim 10, wherein the fusion protein comprises an affinity tag or a refolding aid moiety.

29. The method according to claim 10, wherein the additional moieties is selected from the group consisting of His-tag, SlyD, oligo amino acid stretches composed of either positive or negative charged moieties, Strep-tag and FLAG-tag.

30. The method according to claim 12, wherein the fusion protein is at least partially purified between step (b) and step (c) by affinity chromatography or affinity precipitation.

31. The method according to claim 13, wherein in step (b) is performed in the presences of guanidinium hydrochloride at a concentration of more than 3 M.

32. The method according to claim 31, wherein in step (b) is performed in the presence of guanidinium hydrochloride at a concentration of more than 3.75 M.

33. The method according to claim 14, wherein in step (c) is performed in the presence of guanidinium hydrochloride at a concentration of from 1 to 2 M.

34. The method according to claim 10, wherein the fusion protein is purified between step (b) and step (c).

35. The method according to claim 11, wherein the fusion protein is purified between step (b) and step (c) by affinity purification.

36. The method according to claim 12, wherein the fusion protein is purified between step (b) and step (c) by affinity chromatography.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.

(2) The present invention is further described by the following examples and the drawing figures, yet without being restricted thereto.

(3) FIG. 1: SDS-PAGE of refolding of 21N.sup.pro (HoBi)-pep6His in five different refolding buffers containing 2.5M urea. 100 l refolding batch (c=200 g/ml) were centrifuged at 20,000g. The supernatant was precipitated by TCA. Pellet and supernatant were resuspended in 10 l 1 Magic Mix sample buffer and loaded onto NuPAGE Bis-Tris 4-12% gels. As control the same amount of inclusion body was precipitated and analyzed. M . . . PageRuler Prestained Protein Ladder, P . . . pellet, S . . . supernatant, co . . . control, b . . . buffer. Amount of protein in PAGE samples: P and S together 20 g.

(4) FIG. 2: Refolding efficiencies of D21N.sup.pro (HoBi)-pep6His in five different buffers in presence of 2.5M Urea. Gel was scanned in and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.

(5) FIG. 3: Refolding of 21N.sup.pro (HoBi)-pep6His in 500 mM NaCl, mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol with increasing Urea concentrations up to 4.5M. 100 l refolding batch (c=200 g/ml) were centrifuged at 20,000g. The supernatant was precipitated by TCA. Pellet and supernatant were resuspended in 10 l 1 Magic Mix sample buffer and loaded onto NuPAGE Bis-Tris 4-12% gels. As control the same amount of inclusion body was precipitated and analyzed. M . . . PageRuler Prestained Protein Ladder, P . . . pellet, S . . . supernatant, co . . . control. Amount of protein in PAGE samples: P and S together 20 g.

(6) FIG. 4: Refolding efficiencies of 21N.sup.pro (HoBi)-pep6His in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol with increasing Urea concentrations up to 4.5M. Gel was photographed and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.

(7) FIG. 5: Refolding of 21N.sup.pro (HoBi)-pep6His in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol containing 3M (A), 3.5M (B) and 4M (C) Urea. Each time point 100 l sample were extracted from the refolding batch (c=200 g/ml) and precipitated by TCA. After resuspension in 10 l 1 Magic Mix sample buffer samples were onto NuPAGE Bis-Tris 4-12% gels. M . . . PageRuler Prestained Protein Ladder, P . . . pellet, S . . . supernatant, co . . . control, b . . . buffer. Amount of protein in each PAGE sample: 10 g (A and C) and 5 g (B).

(8) FIG. 6: Determination of the refolding kinetic of 21N.sup.pro (HoBi)-pep6His in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol in presence of 3M, 3.5M and 4M Urea. Gels were photographed and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.

(9) FIG. 7: SDS-PAGE of refolding of 21N.sup.pro (HOBi)-SDD-diUbi-pep6His in five different refolding buffers containing 2.5M urea. 100 l refolding batch (c=200 g/ml) were centrifuged at 20,000g. The supernatant was precipitated by TCA. Pellet and supernatant were resuspended in 10 l 1 Magic Mix sample buffer and loaded onto NuPAGE Bis-Tris 4-12% gels. As control the same amount of inclusion body was precipitated and analyzed. M . . . PageRuler Prestained Protein Ladder, P . . . pellet, S . . . supernatant, co . . . control, b . . . buffer. Amount of protein in PAGE samples: P and S together 20 g.

(10) FIG. 8: Refolding of 21N.sup.pro (HoBi)-SDD-diUbi-pep6His in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol with increasing Urea concentrations up to 4.5M. 100 l refolding batch (c=200 g/ml) were centrifuged at 20,000g. The supernatant was precipitated by TCA. Pellet and supernatant were resuspended in 10 l 1 Magic Mix sample buffer and loaded onto NuPAGE Bis-Tris 4-12% gels. As control the same amount of inclusion body was precipitated and analyzed. M . . . PageRuler Prestained Protein Ladder, P . . . pellet, S . . . supernatant, co . . . control. Amount of protein in PAGE samples: P and S together 20 g.

(11) FIG. 9: Fermentation of. D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4) and D21EDDIE-MCP-1 (SEQ.ID.NO. 5): Course of formation of biomass (DCW), titer of the fusion proteins and titer of MCP-1 (SEQ ID.NO. 10) within the fusion proteins

(12) x-axis stands for the feed time in [h]

(13) left y-axis stands for the titer in [g/L]

(14) right y-axis stands for the DCW (dry cell weight) in [g/L]

(15) A: DCW of cells producing D21EDDIE-MCP-1 (SEQ.ID.NO. 5)

(16) B: DCW of cells producing D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4)

(17) C: titer of D21EDDIE-MCP-1 (SEQ.ID.NO. 5)

(18) D: titer of D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4)

(19) E: titer of MCP-1 within D21EDDIE-MCP-1 (SEQ.ID.NO. 5)

(20) F: titer of MCP-1 within D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4)

(21) FIG. 10: Fermentation of. D21N.sup.pro(HoBi)-MCP-1 (SEQ.ID.NO. 4) and D21EDDIE-MCP-1 (SEQ.ID.NO. 5): Comparison of the titer of the fusion proteins, the titer of MCP-1 within the fusion proteins and the specific titer of MCP-1

(22) Left y-axis stands for the titer in [g/L]

(23) Right y-axis stands for the specific titer in [mg/g DCW]

(24) EDDIE stands for D21EDDIE-MCP-1 (SEQ.ID.NO. 5)

(25) HoBi stands for D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4)

(26) a: titers of the fusion proteins

(27) b: titers of MCP-1 within the fusion proteins

(28) c: specific titer of MCP-1

(29) FIG. 11: Expression of N.sup.pro-fusion proteins D21Npro (HoBi)-pep6His (SEQ.ID.NO. 6), D21Npro (HoBi)-SOD-FLS (SEQ.ID.NO. 8), D21EDDIE-pep6His (SEQ.ID.NO. 7) and D21EDDIE-SOD-FLS (SEQ.ID.NO. 9): SDS-PAGE of soluble (S) and insoluble (IB) fractions three hours after induction (20 mol IPTG/g CDM).

(30) Lane 1: molecular weight marker

(31) Lane 2: D21Npro (HoBi)-pep6His (SEQ.ID.NO. 6), S

(32) Lane 3: D21Npro (HoBi)-pep6His (SEQ.ID.NO. 6), IB

(33) Lane 4: D21Npro (HoBi)-SOD-FLS (SEQ.ID.NO. 8), S

(34) Lane 5: D21Npro (HoBi)-SOD-FLS (SEQ.ID.NO. 8), IB

(35) Lane 6: D21EDDIE-SOD-FLS (SEQ.ID.NO. 9), S

(36) Lane 7: D21EDDIE-SOD-FLS (SEQ.ID.NO. 9), IB

(37) FIG. 12: Refolding and Cleavage kinetic for D21Npro (HoBi)-SOD-FLS (SEQ.ID.NO. 8)(A) and D21EDDIE-SOD-FLS (SEQ.ID.NO. 9)(B) after solubilization in 8 M Urea and refolding in Tris buffer at a protein concentration of c=0.1 mg/L in presence of increasing concentration of residual Urea.

(38) x-axis stands for time in [min]

(39) y-axis stands for cleavage yield in [%]

(40) A: cleavage yield in [%] at 0.4 M Urea

(41) B: cleavage yield in [%] at 1 M Urea

(42) C: cleavage yield in [%] at 2 M Urea

(43) D: cleavage yield in [%] at 3 M Urea

EXAMPLES

(44) Materials and Methods

(45) Protein Expression

(46) 21N.sup.pro (HOBi (SEQ.ID.No.1); deletion of amino acids 2 to 21 of (SEQ.ID.No.2)) was cloned into pET30a vectors harboring pep6His as well as SDD-diUbi-pep6His as target proteins using NdeI and SpeI. The vectors were transformed into E. coli BL21 (DE3) by electroporation and cells were grown over night at 37 C. Cells were diluted 1:100 and incubated at 37 C. until OD.sub.600 reached 0.5. Protein expression was induced by addition of 1M IPTG (isopropyl -D-1-thiogalactopyranoside) to a final concentration of 1 mM IPTG followed by an incubation for four hours at 37 C. Cells were harvested by centrifugation. Lysis was carried out using a french press. Inclusion bodies were harvested by a further centrifugation step.

(47) TABLE-US-00001 21N.sup.Pro(HoBi)autoproteasemoiety (SEQ.ID.NO.1) MEPLYDKNGAVLFGEPSDTHPQSTLKLPHPRGEKEVIVGI RDLPRKGDCRTGNRLGPVSGLFVKPGPVFYQDYSGPVYHR APLEQFKQAPMCEVTKRIGRVTGSDGNLYHMYVCTDGCIL VKTAKREGQDVLKWVYNVLDSPIWVTSC
The SVDKLAAALEHHHHHH motif (SEQ.ID.No.3) is a model peptide attached to the autoprotease moiety.

(48) TABLE-US-00002 N.sup.Pro(HoBi)autoproteasemoiety (SEQ.ID.NO.2) MELLNFELLYKTYKQKPAGVQEPLYDKNGAVLFGEPSDTH PQSTLKLPHPRGEKEVIVGIRDLPRKGDCRTGNRLGPVSG LFVKPGPVFYQDYSGPVYHRAPLEQFKQAPMCEVTKRIGR VTGSDGNLYHMYVCTDGCILVKTAKREGQDVLKWVYNVLD SPIWVASC
Refolding in Different Buffers in Presence of 2.5M Urea

(49) Inclusion bodies were solubilized in 8M Urea/50 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5/10 mM MTG and refolded in

(50) a) buffer 1: 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol

(51) b) buffer 2: 1M Tris/HCl pH 7.5, 5% glycerol

(52) c) buffer 13: 300 mM Arg/HCl, 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 9.0, 5% glycerol

(53) d) buffer 17: 100 mM NaCl, 100 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol

(54) e) buffer 19: 150 mM NaCl, 50 mM NaPhosphate pH 7.5, 5% glycerol

(55) d) buffer 23: 150 mM NaCl, 20 mM Tris/HCl pH 7.5, 5% glycerol 8M Urea/50 mM (NH.sub.4).sub.2HPO.sub.4 pH7.5 was added to a final concentration of 2.5M. Refolding was stopped after 68 hours at 20 C. by centrifugation at 20,000g. The supernatant was precipitated by TCA. Pellet and supernatant were re-suspended in a gel loading buffer containing 8M urea to prevent further refolding in sample buffer and analyzed by gel electrophoresis using Bis-Tris gels. After staining and destaining, gels were photographed and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.
Refolding in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% Glycerol Under Various Urea Concentrations

(56) Inclusion bodies were solubilized in 8M Urea/50 mM (NH.sub.4).sub.2HPO.sub.4 pH7.5/10 mM MTG (Methylthioglycerole) and refolded in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol. 8M Urea/500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol was added to the refolding batch to final concentrations of 2.5, 3, 3.5, 4 and 4.5 M Urea. Refolding was stopped after 44 h at 20 C. by centrifugation at 20,000g for 15 minutes and room temperature and were resuspended in a gel loading buffer containing 8M urea to prevent further refolding in sample buffer and analyzed by gel electrophoresis using Bis-Tris gels. After staining and destaining, gels were photographed and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.

(57) Determination of the Refolding Kinetics in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% Glycerol Under Three Different Urea Concentrations

(58) Inclusion bodies were solubilized in 8M Urea/500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH7.5, 5% glycerol/10 mM MTG and refolded in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol at 20 C. 8M Urea/500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol was added to the refolding batch to final concentrations of 3, 3.5 and 4 M Urea. After defined time points (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 and 30 hours) samples were extracted, precipitated by TCA (trichloroacetic acid) and resuspended in a gel loading buffer containing 8 M Urea to eliminate further refolding in sample buffer. Samples were loaded onto Bis-Tris gels. After staining and destaining gels were photographed and band intensities were determined by densitometry using an AlphaDigDoc 1200 instrument.

Example 1: 21Npro (HoBi)-pep6His

(59) Refolding in Different Buffers in Presence of 2.5M Urea

(60) Refolding studies revealed that 21N.sup.pro (HoBi) was able to cleave off pep6His in presence of 2.5 M urea in all buffers with different efficiencies (FIG. 1 and FIG. 2).

(61) Refolding in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% Glycerol Under Various Urea Concentrations

(62) SDS-PAGE shows that 21N.sup.pro (HoBi) was able to cleave off pep6His at 4.5M urea (FIG. 3). Graph in FIG. 4 illustrates solubility and refolding efficiencies of 21N.sup.pro (HoBi)-pep6His under different urea concentrations. It can be assumed that the higher the urea concentration the lower the refolding efficiency.

(63) Determination of the Refolding Kinetics in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% Glycerol Under Three Different Urea Concentrations

(64) FIG. 5 shows the refolding kinetic of 21N.sup.pro (HoBi)-pep6His in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% glycerol at 3, 3.5 and 4M Urea. The higher the urea concentration, the slower the cleavage reaction took place (FIG. 6).

Example 2: 21Npro (HOBi)-SDD-diUbi-pep6His

(65) Refolding in Different Buffers in Presence of 2.5M Urea

(66) Analysis of refolding behavior of 21N.sup.pro (HoBi)-SDD-diUbi-pep6His by SDS-PAGE showed that 21N.sup.pro (HoBi) was able to cleave off SDD-diUbi-pep6His in presence of 2.5 M urea in majority of the buffers (except buffer 2 and buffer 13) with different efficiencies (FIG. 7). N-terminal sequencing determined that the band already visible in the control lane of 21N.sup.pro (HoBi)-SDID-diUbi-pep6His at the molecular weight of approx. 18 kD derived from in vivo cleaved 21N.sup.pro (HoBi). Determination of the refolding efficiency was left out of account due to the rather broad band of SDD-diUbi-pep6His.

(67) Refolding in 500 mM NaCl, 20 mM (NH.sub.4).sub.2HPO.sub.4 pH 7.5, 5% Glycerol Under Various Urea Concentrations

(68) SDS-PAGE in FIG. 8 illustrates that 21N.sup.pro (HoBi)-SDD-diUbi-pep6His showed still cleavage activity 4.5M urea. Refolding efficiencies of 21N.sup.pro (HoBi)-SDD-diUbi-pep6His were not calculated due to the rather broad band of the target protein SDD-diUbi-pep6His but it is visible that refolding efficiency decreases with raising urea concentration.

Example 3: Expression of D21Npro (HOBi)-MCP-1, SEQ.ID.NO. 4, and D21EDDIE-MCP-1, SEQ.ID.NO. 5 with E. coli in Fed Batch Mode

(69) Generation of Bacterial Strains and Description of Recombinant Proteins

(70) Molecular Weights

(71) MCP-1 (SEQ.ID.NO. 10): 8.7 kD

(72) D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4): 25.2 kD

(73) D21EDDIE-MCP-1 (SEQ.ID.NO. 5): 25.3 kD

(74) Sequences

(75) TABLE-US-00003 D21N.sup.pro(HoBi)-MCP-1(SEQ.ID.NO.4): MEPLYDKNGAVLFGEPSDTHPQSTLKLPHPRGEDEVEVGIRDLPRKGDCRTGNRLGPVSGLFVK PGPVFYQDYSGPVYHRAPLEQFKQTPMEETTKRIGRVTGSDGNLYHMYVETDGEILVKQAKREG QDVLKWTYNTLDSPIWVTSCQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFK TIVAKEICADPKQKWVQDSMDHLDKQTQTPKT D21EDDIE-MCP-1(SEQ.ID.NO.5): MEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETTLRDLPRKGDCRSGNHLGPVSGIYIK PGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRVTGSDGKLYHIYVEVDGEILLKQAKRGT PRTLKWTRNTTNCPLWVTSCQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFK TIVAKEICADPKQKWVQDSMDHLDKQTQTPKT MCP-1(SEQ.ID.NO.10): QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSM DHLDKQTQTPKT

(76) TABLE-US-00004 TABLE 1 List of generated expression strains Protein Strain Plasmid Promoter D21N.sup.pro(HoBi)-MCP-1 B121 (DE3) pET30a(+) T7 (SEQ. ID. NO. 4) Novagen # 6950 from Novagen D21EDDIE-MCP-1 B121 (DE3) pET30a(+) T7 (SEQ. ID. NO. 5) Novagen # 6950 from Novagen

(77) Codon optimized genes of interest (GOIs) were synthesized (Geneart AG, Germany) and provided in the Geneart plasmids with an NdeI restriction site and an EcoRI restriction site as well as 2 TAA stop codons. The GOI DNAs obtained were digested with NdeI and EcoRI, and ligated into a pET30a vector (Novagen, Merck Millipore) restricted with the same enzymes.

(78) For expression of Npro fragments E. coli strain BL21 (DE3) was used. E. coli BL21 (DE3) was transformed and grown o/n at 37 C.

(79) 3 clones were picked, resuspended in Luria-Bertani (LB) medium (Bertani, et al. 1951), grown at 37 C. until an optical density (OD550 nm) 0.2-0.5 and frozen as glycerol stock, all separately.

(80) For expression evaluation clone screenings were performed: LB medium was inoculated from glycerol stock and grown overnight at 37 C. For the clone screening production culture LB medium was inoculated with a seed culture to an OD of 0.2, grown until OD 1. Subsequently, protein expression was induced by addition of 1 mM IPTG (Isopropyl -D-1-thiogalactopyranoside) to the suspension. After incubation for 4 hours at 37 C., the cell pellets were harvested by centrifugation, stored at 20 C. and analyzed for productivity by SDS (Sodium Dodecyl sulfate) Page (Polyaclyamide gel electrophoresis) as follows: Sample preparation: BugBuster Extraction Reagent (Novagen-Merck) plus Lysonase Bioprocessing Reagent mix (Novagen-Merck) Gel: NuPage 12% Bis Tris, 1.0 mm (Invitrogen) Sample buffer: NuPAGE LDS Sample Buffer (Invitrogen) Reducing agent: 2-Mercaptoethanol (Sigma-Aldrich) Running buffer: NuPAGE MES SDS Running Buffer (Invitrogen) Detection: SimplyBlue SafeStain (Invitrogen)
Cultivation Mode and Process Analysis
Preculture

(81) 300 mL preculture medium (media composition as described in Example 4) in a glass shake flask were inoculated from a glycerol stock and grown until an optical density (OD550 nm) of 1.25 to 2.75 is reached.

(82) Main Culture

(83) The cells were grown in a 7 L (5 L net volume, 3 L batch volume) computer-controlled bioreactor (Sartorius Stedim Biotech AG, Germany) equipped with standard control units. The batch medium is described in Example 4 under Media Composition. The pH is maintained at a set-point of 6.80.2 by addition of 25% ammonia solution (Merck), the temperature is set to 37 C.0.5 C. and the aeration rate was fixed 5 L per minute. In order to avoid oxygen limitation, the dissolved oxygen level was stabilized above 20% saturation by stirrer speed and by enrichment with pure oxygen. The content of O.sub.2 and CO.sub.2 in the outlet air was determined by a photoacoustic multi-gas analyzer (Innova). Foaming was suppressed by addition of antifoam suspension (PPG 2000, Dow). For inoculation 30 ml preculture were transferred aseptically to the bioreactor. Feeding was started when the culture entered stationary phase. Fed-Batch regime with an exponential substrate feed was used to provide a constant growth rate of 0.25 h.sup.1 for 8.5 hours and afterwards the feed was changed to a constant mode at the actual feed rate. The feed medium is described in Example 4 under Media Composition

(84) Induction

(85) Induction was performed in a conventional mode by a single pulse directly into the bioreactor. The supplied amount of Isopropyl -D-1-thiogalactopyranoside (IPTG) was calculated to set a concentration of 1 mM IPTG at the end of the process in order to gain a fully induced system.

(86) Offline Analysis

(87) Optical density (OD) was determined with a Genesis 10 spectrophotometer from Spectronic Instruments, USA, at 550 nm. The OD550 nm was measured in a range of 0.2-0.6. Above an extinction of 0.6 the samples were appropriately diluted with OD-buffer (20.7 g/L Na.sub.2HPO.sub.4*12H.sub.2O, 5.7 g/L KH.sub.2PO.sub.4 and 11.6 g/L NaCl). Comparable culture broth dilutions as the measured OD-samples were filtered (pore size 0.2 m) to generate blank values.

(88) Bacterial dry matter (dry cell weights, DCW) was determined by centrifugation of 10 ml of the cell suspension, re-suspension in distilled water followed by centrifugation, and again re-suspension. Then the cell dry weight was determined using a Moisture analyzer ((Sartorius Stedim Biotech AG, Germany). Samples of 35 g culture broth were centrifuged. The supernatant was discarded, residual liquid removed and the weight of the biomass pellet determined.

(89) Glucose in the culture supernatant was measured with the Glucose Analyzer YSI 2700 Select (Yellow Springs). The content of recombinant protein is determined by reducing SDS Page as follows: Sample preparation: BugBuster Extraction Reagent (Novagen-Merck) plus Lysonase Bioprocessing Reagent mix (Novagen-Merck) Gel: NuPage 12% Bis Tris, 1.0 mm (Invitrogen) Sample buffer: NuPAGE LDS Sample Buffer (Invitrogen) Reducing agent: 2-Mercaptoethanol (Sigma-Aldrich) Running buffer: NuPAGE MES SDS Running Buffer (Invitrogen) Detection: SimplyBlue SafeStain (Invitrogen)
Results

(90) Both fusion proteins, D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4) and D21EDDIE-MCP-1 (SEQ.ID.NO. 5), were expressed as insoluble Inclusion Bodies (IB) (FIGS. 9 and 10)

(91) Summary:

(92) FIGS. 9 and 10 clearly show, that the titer for MCP-1 (SEQ. ID. NO. 10) alone was significantly increased by expressing D21N.sup.pro (HoBi)-MCP-1 (SEQ.ID.NO. 4) compared to D21EDDIE-MCP-1 (SEQ.ID.NO. 5). The volumetric (g of MCP-1 per liter of culture broth) as well as the specific (mg of MCP-1 per g of cell dry weight, DCW) titer were increased 1.5- and 1.4-fold respectively

Example 4: Refolding and Cleavage of Npro (HoBi) Fusion Proteins Compared to EDDIE Fusion Proteins with pep6His (SEQ.ID.NO. 11) and SOD-FLS (SEQ.ID.NO.12) as Fusion Partner

(93) Generation of Bacterial Strains and Description of Recombinant Proteins

(94) Experiments were performed with the B-strain BL21(DE3) provided by Novagen. The designation (DE3) indicates that the hosts were lysogens of DE3 prophage, carrying a chromosomal copy of the T7 RNA polymerase gene under control of the lacUV5 promoter, making these strains suitable for protein expression using T7 or T7 lac promoters (Studier and Moffat, 1986; Studier et al., 1990). Expression of target proteins were performed with pET30a plasmid from Novagen (pET System manual, 11.sup.th edition).

(95) TABLE-US-00005 TABLE 2 List of generated expression strains Protein Strain Plasmid Promoter D21Npro(HoBi)- BL21 (DE3) pET30a T7 pep6His (SEQ. ID. NO. 6) D21Npro(HoBi)-SOD- BL21 (DE3) pET30a T7 FLS (SEQ. ID. NO. 8) D21EDDIE-pep6His BL21 (DE3) pET30a T7 (SEQ. ID. NO. 7) D21EDDIE-SOD-FLS BL21 (DE3) pET30a T7 (SEQ. ID. NO. 9)

(96) TABLE-US-00006 D21Npro(HoBi)-pep6His (SEQ.ID.NO.6) MEPLYDKNGAVLFGEPSDTHPQSTLKLPHPRGEKEVIVGIRDLPRKGDCR TGNRLGPVSGLFVKPGPVFYQDYSGPVYHRAPLEQFKQAPMCEVTKRIGR VTGSDGNLYHMYVCTDGCILVKTAKREGQDVLKWVYNVLDSPIWVASCSV DKLAAALEHHHHHH D21Npro(HoBi)-SOD-FLS (SEQ.ID.NO.8) MEPLYDKNGAVLFGEPSDTHPQSTLKLPHPRGEDEVEVGIRDLPRKGDCR TGNRLGPVSGLFVKPGPVFYQDYSGPVYHRAPLEQFKQTPMEETTKRIGR VTGSDGNLYHMYVETDGEILVKQAKREGQDVLKWTYNTLDSPIWVTSCSV MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GTAQVDDYKDDDDKGGGGSGGGGSWSHPQFEK D21EDDIE-SOD-FLS (SEQ.ID.NO.9) MEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETTLRDLPRKGDCR SGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGR VTGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWTRNTTNCPLWVTSCDT MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GTAQVDDYKDDDDKGGGGSGGGGSWSHPQFEK D21EDDIE-pep6His (SEQ.ID.NO.7) MEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETTLRDLPRKGDCR IYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRVTGSDGKLYH IYVEVDGEILLKQAKRGTPRTLKWTRNTTNCPLWVTSCDTSVDKLAAALEHHHHHH Pep6His (SEQ.ID.NO.11) SVDKLAAALEHHHHHH SOD-FLS (SEQ.ID.NO.12) SVMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GTAQVDDYKDDDDKGGGGSGGGGSWSHPQFEK
Cultivation Mode and Process Analysis

(97) The cells were grown in a 10 L (5 L working volume) computer-controlled bioreactor (MBR; Wetzikon, CH) equipped with standard control units. The pH is maintained at a set-point of 7.00.05 by addition of 25% ammonia solution (ACROS Organics), the temperature was set to 37 C.0.5 C. In order to avoid oxygen limitation, the dissolved oxygen level was stabilized above 30% saturation by stirrer speed and aeration rate control. The content of O.sub.2 and CO.sub.2 in the outlet air was determined by a Hartmann and Braun Advanced Optima gas analyzer. Dielectric capacity and conductivity were measured with the Biomass monitor, model 214M (Aber Instruments, Aberystwyth, UK) set. Foaming was suppressed by addition of antifoam suspension (PPG2000, Bussetti, Vienna) with a concentration of 0.5 ml/l feed medium. For inoculation, a deep frozen (80 C.) working cell bank vial, was thawed and 1 ml (optical density OD.sub.600=1) was transferred aseptically to the bioreactor. Feeding is started when the culture, grown to a bacterial dry matter of 7.5 g in 4.0 L batch medium, entered stationary phase. Fed-Batch regime with an exponential substrate feed was used to provide a constant growth rate of 0.2 h.sup.4 during 2 doubling times. The substrate feed was controlled by increasing the pump speed according to the exponential growth algorithm, x=x.sub.o.Math.e.sup.t, with superimposed feedback control of weight loss in the substrate tank (Cserjan-Puschmann et al., 1999). The feed medium provided sufficient components to yield 129 g of bacterial dry matter.

(98) Induction

(99) Induction was performed in a conventional mode by a single pulse directly into the bioreactor. The supplied amount of IPTG was calculated to set a concentration of 20 mol IPTG/g CDMat the end of the process in order to gain a fully induced system.

(100) Media Composition

(101) The minimal medium used in this study contained 3 g KH.sub.2PO.sub.4 and 6 g K.sub.2HPO.sub.4*3H.sub.2O per liter. These concentrations provide the required buffer capacity and serve as P and K source as well. The other components were added in relation of gram bacterial dry matter to be produced: sodium citrate (trisodium salt*2H.sub.2O; ACROS organics) 0.25 g, MgSO.sub.4*7H.sub.2O 0.10 g, CaCl.sub.2*2H.sub.2O 0.02 g, trace element solution 50 l and glucose*H.sub.2O 3 g. To accelerate initial growth of the population, the complex component yeast extract 0.15 g is added to the minimal medium to obtain the batch medium. For the feeding phase 1 L of minimal medium are prepared according to the amount of biological dry matter 129 g to be produced in the feeding phase, whereby P-salts are again added per liter. Trace element solution: prepared in 5 N HCl (g/L): FeSO.sub.4*7H.sub.2O 40.0, MnSO.sub.4*H.sub.2O 10.0, AlCl.sub.3*6H.sub.2O 10.0, CoCl.sub.2 (Fluka) 4.0, ZnSO.sub.4*7H.sub.2O 2.0, Na.sub.2MoO.sub.2*2H.sub.2O 2.0, CuCl.sub.2*2H.sub.2O 1.0, H.sub.3BO.sub.3 0.50.

(102) Offline Analysis

(103) Optical density (OD) was measured at 600 nm. Bacterial dry matter was determined by centrifugation of 10 ml of the cell suspension, re-suspension in distilled water followed by centrifugation, and re-suspension for transfer to a pre-weighed beaker, which was then dried at 105 C. for 24 h and re-weighed. The progress of bacterial growth was determined by calculating the total amount of biomass (total bacterial dry matter BDM; also termed cell dry weight CDW).

(104) The quantification of the expressed fusion protein as illustrated in FIG. 11 was performed with SDS-PAGE by means of a linear regression curve of a reference. Therefore, the samples of solubilized IBs were diluted to be within the calibration range and protein content of bands was determined densitometrically by the ImageQuantTL Software.

(105) Expression systems listed in Table 2 are used to produce the fusion proteins of D21N.sup.pro (Hobi) and D21EDDIE with pep6His (SEQ.ID.NO. 11) and SOD-FLS (SEQ.ID.NO. 12 as fusion partners to compare the cleavage properties of the two autoprotease variants under different refolding conditions. Induction is performed as single pulse one doubling past feed start in order to gain a fully induced system.

(106) TABLE-US-00007 TABLE 3 Production of D21N.sup.pro(HoBi)-pep6His (SEQ ID No. 6), D21N.sup.pro(HoBi)-SOD-FLS (SEQ ID No. 8), D21EDDIE-pep6His (SEQ ID No. 7), and D21EDDIE-SOD-FLS (SEQ ID No. 9) D21N.sup.pro 21N.sup.pro 21EDDIE- 21EDDIE- (HoBi)- (HoBi)- pep6His SOD-FLS pep6His SOD-FLS SEQ ID SEQ ID SEQ ID NO. 6 SEQ ID No. 8 No. 7 NO. 9 Total 126 123 131 131 yield of bacterial dry matter (DCW) [g] Cserjan-Puschmann, M.; Kramer, W.; Drrschmid, E.; Striedner, G.; Bayer, K. Metabolic approaches for the optimisation of recombinant fermentation processes. Appl. Microbiol. Biotechnol, 1999, 53, 43-50. Studier, F. W., and Moffatt, B. A. Use of the bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol., 1986, 189, 113-130. Studier, F. W.; Rosenberg, A. L.; Dunn, J. J.; Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Meth. Enzym., 1990, 185, 60-89.
Preparation of Inclusion Bodies (IB)

(107) The IBs of the above mentioned proteins were harvested as described previously (Walter et al., 2013). The wet cell paste was harvested using a disk centrifuge (Pathfinder PSC 1-06-177; GEA GEA Westfalia Separator Group, Oelde, Germany) and re-suspended in 50 mM Tris, 50 mM NaCl, and 0.02% Tween at pH 8.0 using an ultra turrax (IKA, Staufen, Germany) to obtain a dry matter concentration of 30 g/L. The slurry was passed twice through a Panda 2K homogenizer (GEA Niro Soavi S.p.A., Italy) at a pressure of 1000 bar. IBs were separated using a disk centrifuge, and the resulting pellet was washed twice with 20 mM Tris, 0.5 M NaCl, and 0.02% Tween at pH 8.0. After centrifugation, the pellet was re-suspended using the ultra turrax and washed once with 0.5 M NaCl. After each washing step, the IBs were separated using the disk centrifuge. The final pellet was re-suspended in water to obtain a 40% IB suspension and stored at 20 C. Prior to the experiments, the IBs were lyophilized and stored at 4 C. C. Walther, S. Mayer, A. Trefilov, G. Sekot, R. Hahn, A. Jungbauer, A. Drauer, (2013): Prediction of Inclusion Body Solubilzation From Shaken to Stirred Reactors, Biotechnology & Bioengineering, 111: 84-94
Solubilization of Inclusion Bodies (IB)

(108) The IBs solubilization was carried out as described previously (Walther et al., 2013). For solubilization of IB proteins, lyophilized IBs were resuspended in water for 1 h. The IB suspension was dissolved at a ratio of 1:10 in the corresponding solubilization buffer. The concentration of buffer ingredients was adjusted taking the dilution factor of IB suspension into account to achieve resulting urea concentrations of 8 M or 4 M and 5M GuHCl, respectively. Additionally, the solubilization buffers contained final concentration 50 mM Tris and 100 mM MTG at a pH 7.3. After 2 h, solubilization in the reactor was stopped by at 13,200 rpm at 21 C. for 5 min (Centrifuge 5415R, Eppendorf, Germany) and consecutive filtration through 0.22-m filters (Millipore, Billerica, Mass., USA). C. Walther, S. Mayer, A. Trefilov, G. Sekot, R. Hahn, A. Jungbauer, A. Drauer, (2013): Prediction of Inclusion Body Solubilization From Shaken to Stirred Reactors, Biotechnology & Bioengineering, 111: 84-94
Refolding and Cleavage

(109) Refolding was carried out by rapid dilution of the solubilized IBs into refolding buffer in the ratio 1:20 keeping either the residual concentration of the chaotrope constant for all experiments and varying the protein concentration or vice versa. As refolding buffers two compositions were used as listed in Table 4.

(110) TABLE-US-00008 TABLE 4 Buffer compositions used for refolding Name Shortcut Composition pH TRIS buffer Tris 1M Tris, 0.25M sucrose, 2 mM 7.3 EDTA, 20 mM MTG Ammonium Am-Ph 20 mM ammonium phosphate, 5% 7.5 phosphate glycerol, 20 mM MTG buffer
Determination of the Cleavage Yield [%]

(111) The analysis of the cleavage yield was carried out as described previously (Walther et al., 2013). The cleavage yield was determined analyzing aliquots of the refolding samples throughout the renaturation process over time by RP-HPLC. Separation of the fusion protein from the cleaved target and autoprotease enables the calculation of the cleaving yield via the increase of the cleaved target/autoprotease compared to the initial overall fusion protein.

(112) RP-HPLC was performed using a TSKgel Super-Octyl column (4.650/100 mm, 2 m, 110 ) (Tosoh Bioscience, Germany). The buffer system consisted of 0.1% (v/v) trifluoroacetic acid (TFA) in water as buffer A and 0.1% (v/v) TFA in acetonitrile as buffer B. Solubilization and refolding samples were injected directly. Elution was performed using different gradients that were optimized for each fusion protein that was analyzed. Detection proceeded at two wavelengths, 214 nm and 280 nm, to distinguish between proteins and buffer components. Calibration curves were established for all fusion proteins to quantify the protein in solution. C. Walther, S. Mayer, A. Trefilov, G. Sekot, R. Hahn, A. Jungbauer, A. Drauer, (2013): Prediction of Inclusion Body Solubilzation From Shaken to Stirred Reactors, Biotechnology & Bioengineering, 111: 84-94
Results
Cleavage Kinetic of D21N.sup.pro (HoBi)-SOD-FLS (SEQ.ID.NO. 8) Compared to D21EDDIE-SOD-FLS (SEQ.ID.NO. 9) at Different Urea Concentrations:

(113) The IBs were solubilized in 8M Urea and refolded in two different refolding buffers, Tris and AmPh in presence of increasing concentration of Urea. The protein concentration was identical for all experiments, i.e. c=0.1 mg/mL.

(114) FIG. 12 shows that cleavage kinetic and yield is superior for D21N.sup.pro (HoBi)-SOD-FLS (SEQ.ID.NO. 8), when refolded in Tris buffer. The cleavage yield after 24 hours, when refolded in AmPh was also superior for D21N.sup.pro (HoBi)-SOD-FLS (SEQ.ID.NO. 8)(Table 5).

(115) TABLE-US-00009 TABLE 5 Cleavage yield of D21N.sup.pro(HoBi)-SOD-FLS (SEQ. ID. NO. 8)compared to D21EDDIE-SOD-FLS (SEQ. ID. NO. 9) at different Urea concentrations, when refolded in AmPh: D21Npro(HoBi)-SOD-FLS D21EDDIE-SOD-FLS N.sup.pro variant (SEQ. ID. NO. 8) (SEQ. ID. NO. 9) Solubilization 8M Urea/AmPh buffer/ Refolding buffer Residual Urea 0.4 1 2 3 0.4 1 2 3 concentration (M) Cleavage yield 66 21 6 3 7 5 0 0 (%)
Cleavage Yield of D21Npro (HoBi)-pep6His (SEQ.ID.NO. 6) Compared to D21EDDIE-pep6His (SEQ.ID.NO. 7) at Different Urea Concentrations:

(116) The IBs were solubilized in 8M Urea or 5 M GuHCl and refolded in two different refolding buffers, Tris and AmPh in presence of increasing concentration of Urea or GuHCl. The protein concentration was identical for all experiments, i.e. c=0.1 mg/mL.

(117) TABLE-US-00010 TABLE 6 Cleavage yield after 24 hours of D21.sup.Npro(HoBi)-pep6His (SEQ. ID. NO. 6)compared to D21EDDIE-pep6His (SEQ. ID. NO. 7)in Tris buffer or AmPh buffer buffer after solubilization in 8M Urea or 5M GuHCl in presence of increasing concentration of residual chaotrope at the constant protein concentrations of 0.1 mg/mL. D21Npro(HoBi)-pep6His D21EDDIE-pep6His Variant (SEQ. ID. NO. 6) (SEQ. ID. NO. 7) Solubilization/ 8M Urea/Tris Refolding buffer Residual Urea 0.4 1 2 3 0.4 1 2 3 Concentration (M) Cleavage Yield 80 80 64 34 67 55 41 0 (%) after 24 h Solubilization/ 8M Urea/AmPh Refolding buffer Residual Urea 0.4 1 2 3 0.4 1 2 3 Concentration (M) Cleavage Yield prec 68 42 12 n.d. n.d. n.d. n.d. (%) after 24 h Solubilization/ 5M GuHCL/Tris Refolding buffer Residual Urea 0.25 0.5 1 1.5 0.25 0.5 1 1.5 Concentration (M) Cleavage Yield 74 63 23 65 n.d. n.d. n.d. n.d. (%) after 24 h Solubilization/ 5M GuHCL/AmPh Refolding buffer Residual Urea 0.25 0.5 1 1.5 0.25 0.5 1 1.5 Concentration (M) Cleavage Yield 67.5 55 23 prec n.d. n.d. n.d. n.d. (%) after 24 h n.d. . . . not determined
Summary:

(118) The D21N.sup.pro (HoBi) autoprotease activity in increasing concentrations of chaotrop was for both proteins of interest (pep6His, SEQ.ID.NO. 11 and SOD-FLS, SEQ.ID.NO. 12) and in different refolding buffers (FIG. 12, Table 5 and Table 6) superior compared to the D21EDDIE autoprotease.