Investigation of the effect of different culture conditions on recombinant protein production

Abstract

After the COVID-19 pandemic, vaccine production technologies have become the focus of attention of researchers. As a matter of fact, recombinant protein-based antigen production, which is one of them, has taken its place in the first place. Proteins obtained by recombinant DNA technology are used in many industrial areas, especially vaccine applications, due to their reliability. Therefore, it is very important to produce targeted recombinant proteins in large quantities. This study, for the high amounts production of Omp25 protein, which is used as a vaccine candidate against brucellosis, in laboratory conditions, is aimed to reveal the effects of conditions that are the pre-culturing process, inoculation in LB or TB media, denatured or native purification, culturing with/without IPTG. All the results were analyzed by SDS-PAGE, confirmed Western Blot, and the total protein amounts were measured Bradford method. According to the results, Omp25 protein could not be obtained under native purification conditions in both cultures without induction, but it was observed under denatured conditions. This result can be explained that the protein in the cell is either misfolded or incorporated into the membrane. The amount of protein appears to be much higher in the presence of the inducer in both media inoculated with the starter pre-culture compared to the overnight pre-culture; 8.79 mg and 39.4 mg from 1 L culture, respectively. Additionally, as expected, the addition of IPTG increased the amount of protein, approximately one-and-a-half-fold for LB and about three-fold for TB. Finally, it was observed that TB medium provided higher protein production than LB, which can be explained by the presence of glycerol and high yeast extract in the medium. Although our study contains results that will attract the attention of vaccine industry, it should be kept in mind that all process should always be optimized depending on the structure of the targeted protein and thus the production amount can be further increased.

Keywords

• Recombinant protein
• protein amount
• culture condition
• Omp25

Supporting Institution

Yıldız Technical University Scientific Research Projects Coordination Unit.

Project Number

Project number: FBA-2022-4648.

Thanks

The authors are grateful to the Scientific and Technological Research Council of Turkey (TUBITAK, “2247/C STAR COVID-19”) and The Council of Higher Education of the Republic of Turkey (YOK, “YOK100/2000”) for providing the first author a research scholarship. This report involve a part of İlkgül Akmayan's doctoral thesis. This work was supported by Yıldız Technical University Scientific Research Projects Coordination Unit. Project number: FBA-2022-4648. The authors thank Tuğba ATABEY for her contributions to the rOmp25 protein study.

References

1. Anné J, Maldonado B, Van Impe J, Van Mellaert L, Bernaerts K (2012) Recombinant protein production and streptomycetes. J of biotec 158:159-167. doi: 10.1016/j.jbiotec.2011.06.028
2. Atabey T, Acar T, Derman S, Ordu E, Erdemir A, Taşlı P N, Gür G K, Şahin F, Güllüce M, Arasoğlu T (2021) In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis. Protein and Pept Let 28:1138-147. doi:https://doi:10.2174/0929866528666210615104334   Ahmed IM, Khairani-Bejo S, Hassan L, Bahaman AR, Omar RA (2015) Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay. BMC Vet Res 11:275 https://doi.org/10.1186/s12917-015-0587-2
3. Baily G, Krahn J, Drasar B, Stoker N (1992) Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95:271-275.
4. Cloeckaert A, Verger J-M, Grayon M, Zygmunt M S, Grépinet O (1996a) Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene. Infect immun 64:2047-2055. doi: 10.1128/iai.64.6.2047-2055.1996
5. Cloeckaert A, Zygmunt M, Bezard G, Dubray G (1996b) Purification and antigenic analysis of the major 25-kilodalton outer membrane protein of Brucella abortus. Res Microbiol 147:225-235. doi: https://doi.org/10.1016/0923-2508(96)81383-0
6. Coskun KA, Yurekli N, Abay EC, Tutar, M., Al, M, Tutar, Y. (2022). Structure-and Design-Based Difficulties in Recombinant Protein Purification in Bacterial Expression. Protein Detection. 3.
7. Çelebi S, Hacimustafaoğlu M, (2004). Brusellozis. Güncel Pediatri, 2(1), 39-43.
8. Danquah M K, Forde GM (2007) Growth medium selection and its economic impact on plasmid DNA production. J Biosci Bioeng 104:490-497. doi: 10.1263/jbb.104.490

9. Froger A, Hall JE (2007) Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp 6:e253. doi: 10.3791/253
10. Goel D, Bhatnagar R, (2012). Intradermal immunization with outer membrane protein 25 protects Balb/c mice from virulent B. abortus 544. Mol Immun 51: 159 -168. doi:10.1016/j.molimm.2012.02.126
11. Goel D, Rajendran V, Ghosh PC, Bhatnagar R, (2013). Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544. Vaccine, 31(8), 1231–1237. doi:10.1016/j.vaccine.2012.12.04 Hemamalini N, Ezhilmathi S, Mercy AA (2020) Recombinant protein expression optimization in Escherichia coli: A review. Indian J Anim Res 54:653-660. doi: 10.18805/ijar.B-3808
12. Hortsch R, Weuster-Botz D (2011) Growth and recombinant protein expression with Escherichia coli in different batch cultivation media. App microbiol biotechnol 90:69-76. doi: 10.1007/s00253-010-3036-y
13. Hu Z, Chen J-P, Xu J-C, Chen Z-Y, Qu R, Zhang L, Yao W, Wu J, Yang H, Lowrie DB (2022) A two-dose optimum for recombinant S1 protein-based COVID-19 vaccination. Virol J 566:56-59. doi: 10.1016/j.virol.2021.11.011
14. Huleani S, Roberts MR, Beales L, Papaioannou EH (2022) Escherichia coli as an antibody expression host for the production of diagnostic proteins: significance and expression. Crit Rev Biotechnol 42:756-773. https://doi.org/10.1080/07388551.2021.1967871
15. Jiang H, O’Callaghan D, Ding J-B (2020) Brucellosis in China: history, progress and challenge. Infect Dis Poverty 9:101-104. doi: 10.1186/s40249-020-00673-8
16. Khramtsov P, Kalashnikova T, Bochkova M, Kropaneva M, Timganova V, Zamorina S, Rayev M (2021) Measuring the concentration of protein nanoparticles synthesized by desolvation method: Comparison of Bradford assay, BCA assay, hydrolysis/UV spectroscopy and gravimetric analysis. Int J Pharm 599:120422. https://doi.org/10.1016/j.ijpharm.2021.120422
17. Ma QL, Liu AC, Ma XJ, Wang YB, Hou YT, Wang ZH (2015). Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis. Int J Clin Exp Med. 15;8(10):17530-5. PMID: 26770344; PMCID: PMC4694244.
18. Mühlmann M, Forsten E, Noack S, Büchs J (2017) Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Factories 16:1-12. https://doi.org/10.1186/s12934-017-0832-4
19. Naseri N, Mirian M, Mofid MR (2022) Expression of recombinant insulin-like growth factor-binding protein-3 receptor in mammalian cell line and prokaryotic (Escherichia coli) expression systems. Adv Biomed Res 11:19. doi: 10.4103/abr.abr_197_20
20. Overton TW (2014) Recombinant protein production in bacterial hosts. Drug Discov Today 19:590-601. doi: 10.1016/j.drudis.2013.11.008
21. Packiam KAR, Ramanan RN, Ooi CW, Krishnaswamy L, Tey BT (2020) Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches. Appl Microbiol Biotechnol 104:3253-3266. doi.org/10.1007/s00253-020-10454-w
22. Papaneophytou CP, Kontopidis G (2014) Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif 94:22-32. doi: 10.1016/j.pep.2013.10.016
23. Ratnaningsih E, Sukandar SI, Putri RM, Kadja GT, Wenten IG (2022) Optimization of haloacid dehalogenase production by recombinant E. coli BL21 (DE3)/pET-hakp1 containing haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 using Response Surface Methodology (RSM). Heliyon e11546. doi: 10.1016/j.heliyon.2022.e11546
24. Research PW (1998) E. Coli Culture for Protein Expression. In: Investigating the Biochemistry & Cellular Physiology of NHE1EST. [online] Available at. https://home.sandiego.edu/~josephprovost/E.%20Coli%20protein%20expression%20protocol.pdf Accesed: 08 March 2022
25. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172
26. Thongbhubate K, Irie K, Sakai Y, Itoh A, Suzuki H (2021) Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12. AMB Express 11:1-13. https://doi.org/10.1186/s13568-021-01330-5
27. Tiwari AK, Kumar S, Pal V, Bhardwaj B, Rai GP (2011) Evaluation of the recombinant 10-kilodalton immunodominant region of the BP26 protein of Brucella abortus for specific diagnosis of bovine brucellosis. Clin Vaccine Immunol 18:1760-1764. doi: 10.1128/CVI.05159-11
28. Tripathi NK, Babu JP, Shrivastva A, Parida M, Jana AM, Rao PL (2008) Production and characterization of recombinant dengue virus type 4 envelope domain III protein. J Biotechnol 134:278-286. doi: 10.1016/j.jbiotec.2008.02.001
29. Tripathi N K, Shrivastva A, Biswal KC, Rao, PVL (2009). METHODS: Optimization of culture medium for production of recombinant dengue protein in Escherichia coli. Indust. Biotech. 5(3), 179–183. doi:10.1089/ind.2009.3.179
30. Woodward M, Young Jr W, Bloodgood R (1985) Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods 78:143-153. doi: 10.1016/0022-1759(85)90337-0
31. Yao M, Guo X, Wu X, Bai Q, Sun M, Yin D (2022) Evaluation of the Combined Use of Major Outer Membrane Proteins in the Serodiagnosis of Brucellosis. Infect Drug Resist 4093-4100. doi: 10.2147/IDR.S372411
32. Yousefi S, Abbassi-Daloii T, Sekhavati MH, & Tahmoorespur M, (2018). Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen. Microb Pathog 115: 50–56. doi:10.1016/j.micpath.2017.12.045
33. Yousefi S, Tahmoorespur M, Sekhavati MH, (2016) Cloning, expression and molecular analysis ofIranian Brucella melitensis Omp25 gene for designing a subunit vaccine, Res pharm 11(5) 412.doi: 10.4103/1735-5362.192493
34. Yumuk Z, O’Callaghan D (2012). Brucellosis in Turkey—an overview. International J Infect Dis 16(4): e228-e235 Zhang B, Gu H, Yang Y, Bai H, Zhao C, Si M, Su T, Shen X (2019) Molecular mechanisms of AhpC in resistance to oxidative stress in Burkholderia thailandensis. Front Microbiol 10:1483. https://doi.org/10.3389/fmicb.2019.01483
35. Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J (2018) Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Factories 17:1-12. https://doi.org/10.1186/s12934-018-0894-y
36. Zhang Z, Kuipers G, Niemiec Ł, Baumgarten T, Slotboom DJ, de Gier J-W, Hjelm A (2015) High-level production of membrane proteins in E. coli BL21 (DE3) by omitting the inducer IPTG. Microb Cell Factories 14:1-11. https://doi.org/10.1186/s12934-015-0328-z