METHOD FOR PRODUCING A RECOMBINANT BACTERIAL COLLAGEN-LIKE PROTEIN (CLP)

Abstract

A method for producing a recombinant collagen-like protein (CLP) can be performed. The method includes fermenting a host cell, accumulating the CLP in a medium to obtain a fermentation broth, separating the host cell from the fermentation broth to obtain a supernatant, and incubating the supernatant. The CLP can be purified after incubation.

Claims

1. A method for producing a recombinant collagen-like protein (CLP), the method comprising: a) fermenting at least one host cell, expressing a CLP with an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO:1, in a medium, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1, b) accumulating the CLP in the medium, wherein a fermentation broth is obtained, c) separating the at least one host cell from the fermentation broth to obtain a supernatant, d) incubating the supernatant of the fermentation broth of c) for at least 1 h at not more than 25? C. for a folding of the CLP, e) optionally purifying the CLP by at least one selected from the group consisting of: solvent precipitation, tangential flow filtration (TFF), ion exchange chromatography, and reversed-phase chromatography.

2. The method according to claim 1, wherein the amino acid sequence further comprises a deletion of between 38 and 90 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1.

3. The method according to claim 1, wherein the amino acid sequence is at least 60% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

4. The method according to claim 1, wherein the amino acid sequence is at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

5. The method according to claim 1, wherein the CLP is a collagen-like protein from Streptococcus pyogenes.

6. The method according to claim 1, wherein the folding of the CLP in d) is performed at a temperature between ?80? C. and 25? C.

7. The method according to claim 1, wherein the folding of the CLP in d) is performed for a time between 1 h and 48 h.

8. The method according to claim 1, wherein the folding of the CLP in d) is performed with a concentration of the CLP of at least 1 mg/ml.

9. The method according to claim 1, wherein the at least one host cell is a yeast cell or a bacterial cell.

10. The method according to claim 1, wherein the amino acid sequence is 100% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

11. The method according to claim 1, wherein the amino acid sequence is 97% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

12. The method according to claim 1, wherein the amino acid sequence is 98% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

13. The method according to claim 1, wherein the amino acid sequence is 99% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

14. The method according to claim 1, wherein the folding of the CLP in d) is performed at a temperature between 0? C. and 20? C.

15. The method according to claim 1, wherein the folding of the CLP in d) is performed for a time between 1 h and 24 h.

16. The method according to claim 1, wherein the folding of the CLP in d) is performed with a concentration of the CLP of at least 4 mg/ml.

17. The method according to claim 9, wherein the yeast cell is Pichia pastoris.

18. The method according to claim 9, wherein the bacterial cell is E. coli, Corynebacterium or Brevibactetium.

Description

EXAMPLES

[0078] The collagen-like protein was produced in the yeast host cell Pichia pastoris by fermentation. To produce Scl2 from Streptococcus pyogenes in Pichia pastoris, the sequence of the collagen-like protein (full-length protein and truncated variants and no-V-domain variant), has been codon optimized using different algorithms, and cloned in a secretion vector for Pichia pastoris. The sequences used are summarized in SEQ ID NO:1 to SEQ ID NO:9. For each of the specific sequences, a vector was transformed in Pichia pastoris following standard protocol and a standard expression protocol in fed-batch mode was applied (Damasceno, L. M., Huang, C J. & Batt, C. A. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93, 31-39 (2012)). The collagen domain of the Scl2p protein was detected via HPLC analysis in the supernatant of cell culture. Upon fermentation, supernatant has been separated from biomass via centrifugation (12000 g, 5 mins at room temperature).

[0079] The collagen domain of the Scl2p protein based on the sequences SEQ ID NO:1 to SEQ ID NO:9 could be produced under similar conditions using either E. coli, B. choshinensis or C. glutamicum. In case of a production in yeast or C. glutamicum, the collagen domain is secreted by the cell. No cell lysis is needed as an initial purification step in this approach. In case of a production in E. coli a cell lysis is mandatory to remove the collagen domain from the cell.

[0080] The full-length collagen-like protein, a truncated variant (truncation 3) and the no-V-domain variant (based on the gene scl2 from Streptococcus pyogenes) were also expressed in Brevibacillus choshinensis. Therefore, the corresponding DNA sequences were cloned into a suitable secretion vector for B. choshinensis. Transformation of B. choshinensis with the new constructed plasmids was done according to Mizukami et al. 2010 (Curr Pharm Biotechnol 2010, 13:151-258).

[0081] The B. choshinensis strains were analyzed for their ability to produce the different collagen-like proteins in batch cultivations at 33? C. and pH 7 using the DASGIP? parallel bioreactor system from Eppendorf (Hamburg, Germany). The fermentation was performed using 1 L reactors. The production medium (TM medium, Biomed Res Int 2017, 2017: 5479762) contained 10 g/L glucose. Upon fermentation, supernatant has been separated from biomass by centrifugation and was used for SDS PAGE analysis. For all three variants, collagen domain of the Scl2p protein was produced.

[0082] The full-length collagen-like protein and the no-V-domain variant (based on the gene scl2 from Streptococcus pyogenes) were also expressed in Corynebacterium glutamicum. Therefore, the corresponding DNA sequences were cloned together with an upstream located signal peptide for protein secretion into a shuttle vector for C. glutamicum (Biotechnology Techniques 1999, 13: 437-441). The C. glutamicum strain ATCC 13032 was transformed with the new constructed plasmids by means of electroporation as described by Ruan et al. (Biotechnology Letters 2015, 37: 2445-2452).

[0083] The C. glutamicum strains were analyzed for their ability to produce the different collagen proteins in fed-batch cultivations at 30? C. and pH 7 using the DASGIP? parallel bioreactor system from Eppendorf (Hamburg, Germany). The fermentation was performed using 1 L reactors. The production medium contained 20 g/L glucose in the batch phase and the fed-batch phase was run with a glucose feed of 4 g/L*h. Upon fermentation, supernatant has been separated from biomass by centrifugation and was used for HPLC analysis. For both variants, collagen domain of the Scl2p protein was produced. For the truncated variant of the collagen-like protein, titer was higher as for the full-length variant.

[0084] The process steps are summarized below: [0085] 1. Production of collagen-like protein in yeast, E. coli or Corynebacterium [0086] 2. Cell lysis (only for E. coli) [0087] 3. Cell separation (Filtration or centrifugation) [0088] 4. Folding of the CL single strand to form a triple helical structure [0089] 5. Further purification by solvent precipitation and ultrafiltration [0090] 6. Freeze dry of the purified CL protein

[0091] To determine folding kinetics, freeze dried collagen domain of the Scl2p protein coming from a production in Pichia pastoris is dissolved at a concentration of 40 g/L in DI water and unfolded at 40? C. The solution is split up and further diluted in a concentration range from 1-40 g/L. The different samples are then incubated at different temperatures ranging from 4-30? C. for 20 h to obtain a folding kinetic of collagen-like protein in the temperature and concentration range given. The folding rate is determined using Size exclusion chromatography (SEC). The temperature dependent folding of the collagen-like protein is summarized in FIG. 1.

[0092] A second sample set is prepared to cover time dependent folding of the CL protein in a time range of 0.25-1 h and an incubation temperature of 4-20? C. The results are summarized in FIGS. 2 to 4.

Protein Sequences

[0093] SEQ ID NO:1 Streptococcus pyogenes Collagen-like protein (CLP), full length protein [0094] SEQ ID NO:2 Streptococcus pyogenes CLP, truncation 3 [0095] SEQ ID NO:3 Streptococcus pyogenes CLP, truncation 5 [0096] SEQ ID NO:4 Streptococcus pyogenes CLP, no V-domain [0097] SEQ ID NO:5 Streptococcus pyogenes CLP, truncation 5 (AGPR mutant) [0098] SEQ ID NO:6 Streptococcus pyogenes CLP, truncation 5 (QGPR mutant) [0099] SEQ ID NO:7 Streptococcus pyogenes CLP, truncation 5 (VGPA mutant) [0100] SEQ ID NO:8 Streptococcus pyogenes CLP, truncation 5 (SGPR mutant) [0101] SEQ ID NO:9 Streptococcus pyogenes CLP, truncation 5 (VGPK mutant)