Development of therapeutic peptide producers based on Escherichia coli BL21 and their cultivation technology

Introduction. Peptides with a molecular weight of less than 5 kDa have been used in medicine and biotechnology over the past decade for the treatment of various diseases. However, chemical synthesis peptide has several disadvantages, including low yield, reduced efficiency, and high costs. An alternative approach to peptide production is the use of the Escherichia coli expression system. The development of effective peptide synthesis technology remains a critical task because of the low productivity of recombinant strains.

Aim. Developing highly efficient strains of Escherichia coli BL21 expressing therapeutic peptides with a molecular weight of less than 5 kDa in E. coli and their cultivation technology.

Materials and methods. Genetic constructs were obtained using the restriction-ligase method, and their authenticity was confirmed by Sanger sequencing. Cultivation technology was developed using the Design of Experiments approach. The cultivation condition was validated in the Biostat B bioreactor. Hybrid proteins were purified by metal-chelate chromatography, followed by hydrolysis ULP proteas to obtain the target peptides. The quantitative content of the target protein was determined by capillary electrophoresis, and the authenticity of the protein was confirmed by HPLC-MS and ELISA.

Results and discussion. Highly efficient peptide-producing strains were developed. Cultivation conditions were optimized: рН 7.5 ± 0.5, cultivation temperature 37 °C, induction optical density 28 ± 2, IPTG concentration 0.05 мМ. The productivity of the producer strains was up to 4.82 ± 0.05 g/L. Furthermore, samples of the target peptides were isolated and purified.

Conclusion. The productivity of peptides in this study were significantly higher than in previous research. The presented strategy for strain development, cultivation and purification technology can be used production of therapeutic peptides with diverse physical chemicals characteristics in the future.

1. Fosgerau K., Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discovery Today. 2015;20(1):122–128. DOI: 10.1016/j.drudis.2014.10.003.

2. Gerstein H. C., Rutty. C. J. Insulin Therapy: The Discovery That Shaped a Century. Canadian Journal of Diabetes. 2021;45(8):798–803. DOI: 10.1016/j.jcjd.2021.03.002.

3. Lau J.L., Dunn M.K. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorganic & Medicinal Chemistry. 2018;26(10):2700–2707. DOI: 10.1016/j.bmc.2017.06.052.

4. Chandrudu S., Simerska P., Toth I. Chemical methods for peptide and protein production. Molecules. 2013;18(4):4373–4388. DOI: 10.3390/molecules18044373.

5. Mason J. M. Design and development of peptides and peptide mimetics as antagonists for therapeutic intervention. Future Medicinal Chemistry. 2010;2(12):1813–1822. DOI: 10.4155/fmc.10.259.

6. Guzmán F., Barberis S., Illanes A. Peptide synthesis: chemical or enzymatic. Electronic Journal of Biotechnology. 2007;10(2):279–314. DOI: 10.2225/vol10-issue2-fulltext-13.

7. Mueller L. K., Baumruck A. C., Zhdanova H., Tietze A. A. Challenges and perspectives in chemical synthesis of highly hydrophobic peptides. Frontiers in Bioengineering and Biotechnology. 2020;8:162. DOI: 10.3389/fbioe.2020.00162.

8. Ratelade J., Miot M.-C., Johnson E., Betton J.-M., Mazodier P., Benaroudj N. Production of recombinant proteins in the lon-deficient BL21(DE3) strain of Escherichia coli in the absence of the DnaK chaperone. Applied and Environmental Microbiology. 2009;75(11). DOI: 10.1128/AEM.00255-09.

9. Rosano G. L., Ceccarelli E. A. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology. 2014;5:172. DOI: 10.3389/fmicb.2014.00172.

10. Fathi-Roudsari M., Akhavian-Tehrani A., Maghsoudi N. Comparison of Three Escherichia coli Strains in Recombinant Production of Reteplase. Avicenna Journal of Medical Biotechnology. 2016;8(1):16–22.

11. Studier F. W., Daegelen P., Lenski R. E., Maslov S., Kim J. F. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. Journal of Molecular Biology. 2009;394(4):653–680. DOI: 10.1016/j.jmb.2009.09.021.

12. Rodríguez V., Asenjo J. A., Andrews B. A. Design and implementation of a high yield production system for recombinant expression of peptides. Microbial Cell Factories. 2014;13:65. DOI: 10.1186/1475-2859-13-65.

13. Lv G. S., Huo G. C., Fu X. Y. Expression of milk-derived antihypertensive peptide in Escherichia coli. Journal of Dairy Science. 2003;86(6):1927–1931. DOI: 10.3168/jds.S0022-0302(03)73779-5.

14. Li Y., Li X., Wang G. Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expression and Purification. 2006;47(2):498–505. DOI: 10.1016/j.pep.2005.10.022.

15. Rao X., Hu J., Li S., Jin X., Zhang C., Cong Y., Hu X., Tan Y., Huang J., Chen Z., Zhu J., Hu F. Design and expression of peptide antibiotic hPAB-β as tandem multimers in Escherichia coli. Peptides. 2005;26(5):721–729. DOI: 10.1016/j.peptides.2004.12.016.

16. Hay R. T. SUMO: a history of modification. Molecular Cell. 2005;18(1):1–12. DOI: 10.1016/j.molcel.2005.03.012.

17. Mirzadeh K., Shilling P. J., Elfageih R., Cumming A. J., Cui H. L., Rennig M., Nørholm M. H. H., Daley D. O. Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microbial Cell Factories. 2020;19(1):85. DOI: 10.1186/s12934-020-01339-8.

18. Nguyen B.-N., Tieves F., Rohr T., Wobst H., Schöpf F. S., Montoya Solano J. D., Schneider J., Stock J., Uhde A., Kalthoff T., Jaeger K. E., Schmitt L., Schwarz C. Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins. AMB Express. 2021;11(1):48. DOI: 10.1186/s13568-021-01204-w.

19. Wu C.-L., Chih Y.-H., Hsieh H.-Y., Peng K.-L., Lee Y.-Z., Yip B.-S., Sue S.-C., Cheng J.-W. High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli. Biomedicines. 2022;10(6):1351. DOI: 10.3390/biomedicines10061351.

20. Zhou L., Lian K., Wang M., Jing X., Zhang Y., Cao J. The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability. BMC Microbiology. 2022;22(1):220. DOI: 10.1186/s12866-022-02621-y.

21. Ojima-Kato T., Nishikawa Y., Furukawa Y., Kojima T., Nakano H. Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli. Journal of Biological Chemistry. 2023;299(5):104676. DOI: 10.1016/j.jbc.2023.104676.

22. Shafiee F., Rabbani M., Jahanian-Najafabadi A. Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology. Advanced Biomedical Research. 2017;6(1):22. DOI: 10.4103/2277-9175.201334.

23. Shahzadi I., Al-Ghamdi M. A., Nadeem M. S., Sajjad M., Ali A., Khan J. A., Kazmi I. Scale-up fermentation of Escherichia coli for the production of recombinant endoglucanase from Clostridium thermocellum. Scientific Reports. 2021;11(1):7145. DOI: 10.1038/s41598-021-86000-z.

24. Singh S. K., Singh S. K., Tripathi V. R., Khare S. K., Garg S. K. Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate. Microbial Cell Factories. 2011;10:114. DOI: 10.1186/1475-2859-10-114.

25. Uhoraningoga A., Kinsella G. K., Henehan G. T., Ryan B. J. The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production. Bioengineering. 2018;5(4):89. DOI: 10.3390/bioengineering5040089.

26. Larentis A. L., de Cássia Cunha Sampaio H., Martins O. B., Rodrigues M. I., Moitinho Alves T. L. Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design. Journal of Industrial Microbiology & Biotechnology. 2011;38(8):1045–1054. DOI: 10.1007/s10295-010-0879-2.

27. Lessa-Aquino C., Borges Rodrigues C., Pablo J., Sasaki R., Jasinskas A., Liang L., Wunder E. A., Ribeiro G. S., Vigil A., Galler R., Molina D., Liang X., Reis M. G., Ko A. I., Medeiros M. A., Felgner P. L. Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach. PLoS Neglected Tropical Diseases. 2013;7(10):e2499. DOI: 10.1371/journal.pntd.0002499.

28. Lin Y.-P., McDonough S. P., Sharma Y., Chang Y.-F. Leptospira immunoglobulin-like protein B (LigB) binding to the C-terminal fibrinogen αC domain inhibits fibrin clot formation, platelet adhesion and aggregation. Molecular Microbiology. 2011;79(4):1063–1076. DOI: 10.1111/j.1365-2958.2010.07510.x.

29. Latypov V. F., Kornakov I. A., Robustova S. E., Khomutova O. S., Rodionov P. P. Recombinant plasmid DNA pF646, encoding a hybrid polypeptide containing proinsulin asprate, and a strain of Escherichia coli bacteria – a producer of the hybrid polypeptide containing proinsulin aspart. Patent RUS № RU2729353. 2019. Available at: https://patents.google.com/patent/RU2729353C1/ru. Accessed: 16.05.2024. (In Russ.)

30. Casali N., Preston A. E. coli plasmid vectors: methods and applications. Totowa: Humana Press; 2008. 316 p.

31. Ronald M. A. Handbook of Microbiological Media. 2^nd ed. Boca Raton: CRC Press; 1997.

32. Schägger H. Tricine-SDS-PAGE. Nature Protocols. 2006;1(1):16–22. DOI: 10.1038/nprot.2006.4.

33. Umetrics M. User guide to MODDE. 2014.

34. Mandenius C.-F., Brundin A. Bioprocess optimization using design-of-experiments methodology. Biotechnology Progress. 2008;24(6):1191–1203. DOI: 10.1002/btpr.67.

35. Beal J., Farny N. G., Haddock-Angelli T., Selvarajah V., Baldwin G. S., Buckley-Taylor R., Gershater M., Kiga D., Marken J., Sanchania V., Sison A., Workman C. T. Robust estimation of bacterial cell count from optical density. Communications Biology. 2020;3(1):512. DOI: 10.1038/s42003-020-01127-5.

36. Dolnik V. Capillary electrophoresis of proteins 2005–2007. Electrophoresis. 2008;29(1):143–156. DOI: 10.1002/elps.200700584.

37. Marty M. T., Baldwin A. J., Marklund E. G., Hochberg G. K. A., Benesch J. L., Robinson C. V. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical Chemistry. 2015;87(8):4370–4376. DOI: 10.1021/acs.analchem.5b00140.

38. Aucoin M. G., McMurray-Beaulieu V., Poulin F., Boivin E. B., Chen J., Ardelean F. M., Cloutier M., Choi Y. J., Miguez C. B., Jolicoeur M. Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach. Microbial Cell Factories. 2006;5:27. DOI: 10.1186/1475-2859-5-27.

39. Kumar J., Bhat S. U., Rathore A. S. Slow post-induction specific growth rate enhances recombinant protein expression in Escherichia coli: Pramlintide multimer and ranibizumab production as case studies. Process Biochemistry. 2022;114:21–27. DOI: 10.1016/j.procbio.2022.01.009.

40. Donovan R. S., Robinson C. W., Glick B. R. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of thelac promoter. Journal of Industrial Microbiology. 1996;16(3):145–154. DOI: 10.1007/BF01569997.

41. Mühlmann M., Forsten E., Noack S., Büchs J. Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microbial Cell Factories. 2017;16(1):220. DOI: 10.1186/s12934-017-0832-4.

42. Song J. M., An Y. J., Kang M. H., Lee Y.-H., Cha S.-S. Cultivation at 6–10 °C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli. Protein Expression and Purification. 2012;82(2):297–301. DOI: 10.1016/j.pep.2012.01.020.

43. Soleymani B., Mostafaie A. Analysis of Methods to Improve the Solubility of Recombinant Bovine Sex Determining Region Y Protein. Reports of Biochemistry and Molecular Biology. 2019;8(3):227–235.

44. De Mey M., De Maeseneire S., Soetaert W., Vandamme E. Minimizing acetate formation in E. coli fermentations. Journal of Industrial Microbiology & Biotechnology. 2007;34(11):689–700. DOI: 10.1007/s10295-007-0244-2.

45. Warnecke T., Gill R. T. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microbial Cell Factories. 2005;4:25. DOI: 10.1186/1475-2859-4-25.

46. Wang H., Wang F., Wang W., Yao X., Wei D., Cheng H., Deng Z. Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach. PLoS ONE. 2014;9(11):e112777. DOI: 10.1371/journal.pone.0112777.

47. Einsfeldt K., Severo Junior J. B., Corrêa Argondizzo A. P., Medeiros M. A., Moitinho Alves T. L., Almeida R. V., Larentis A. L. Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability. Vaccine. 2011;29(41):7136–7143. DOI: 10.1016/j.vaccine.2011.05.073.

48. Larentis A. L., Monteiro Quintal Nicolau J. F., dos Santos Esteves G., Vareschini D. T., de Almeida F. V. R., Galvão dos Reis M., Galler R., Medeiros M. A. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Research Notes. 2014;7:671. DOI: 10.1186/1756-0500-7-671.

49. Urban A., Ansmant I., Motorin Y. Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae. Journal of Chromatography B. 2003;786(1–2):187–195. DOI: 10.1016/s1570-0232(02)00742-0.

50. Martins Fukuda I., Fidelis Pinto C. F., dos Santos Moreira C., Morais Saviano A., Rebello Lourenço F. Design of Experiments (DoE) applied to Pharmaceutical and Analytical Quality by Design (QbD). Brazilian Journal of Pharmaceutical Sciences. 2018;54: e01006. DOI: 10.1590/s2175-97902018000001006.

51. Kiss B., Gottschalk U., Pohlscheidt M., editors. New Bioprocessing Strategies: Development and Manufacturing of Recombinant Antibodies and Proteins. New York: Springer; 2018.

52. Ladner T., Flitsch D., Lukacs M., Sieben M., Büchs J. Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles. Biotechnology and Bioengineering. 2019;116(12):3215–3227. DOI: 10.1002/bit.27145.

53. Rosano G. L., Ceccarelli E. A. Recombinant protein expression in microbial systems. Frontiers in Microbiology. 2014;5:341. DOI: 10.3389/fmicb.2014.00341.

54. Jiang D.-L., Yao C.-L., Hu N.-J., Liu Y.-C. Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone. Molecules. 2021;26(20):6207. DOI: 10.3390/molecules26206207.

55. Zhao Z.-H., Zhang C.-X., Li J., Zhang A.-Z., Zhao F.-F., Yu G.-P., Jiang N. Effect of tandem repeats of antimicrobial peptide CC34 on production of target proteins and activity of Pichia pastoris. Protein Expression and Purification. 2023;212:106342. DOI: 10.1016/j.pep.2023.106342.