Production of Soluble and Functional Anti-TNF-α Fab' Fragment in Cytoplasm of E. coli: Investigating the Effect of Process Conditions on Cellular Biomass and Protein Yield Using Response Surface Methodology.
Certolizumab
Fab’ antibody fragment
Optimization
Response surface methodology
SHuffle E. coli strain
Journal
The protein journal
ISSN: 1875-8355
Titre abrégé: Protein J
Pays: Netherlands
ID NLM: 101212092
Informations de publication
Date de publication:
10 2021
10 2021
Historique:
accepted:
10
05
2021
pubmed:
24
5
2021
medline:
25
12
2021
entrez:
23
5
2021
Statut:
ppublish
Résumé
With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.In this study, non-glycosylated anti-TNF-α Fab' moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.
Identifiants
pubmed: 34023982
doi: 10.1007/s10930-021-09996-3
pii: 10.1007/s10930-021-09996-3
doi:
Substances chimiques
Recombinant Proteins
0
Certolizumab Pegol
UMD07X179E
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
786-798Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Hua Q et al (2004) Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures. Appl Environ Microbiol 70(4):2354–2366
doi: 10.1128/AEM.70.4.2354-2366.2004
Graumann K, Premstaller A (2006) Manufacturing of recombinant therapeutic proteins in microbial systems. Biotechnol J Healthcare Nutr Technol 1(2):164–186
Georgiou G, Valax P (1996) Expression of correctly folded proteins in Escherichia coli. Curr Opin Biotechnol 7(2):190–197
doi: 10.1016/S0958-1669(96)80012-7
Graham L, Beveridge T, Nanninga N (1991) Periplasmic space and the concept of the periplasm. Trends Biochem Sci 16(9):328–329
doi: 10.1016/0968-0004(91)90135-I
Stock JB, Rauch B, Roseman S (1977) Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem 252(21):7850–7861
doi: 10.1016/S0021-9258(17)41044-1
Wang YY et al (2011) Enhancement of excretory production of an exoglucanase from Escherichia coli with phage shock protein A (PspA) overexpression. J Microbiol Biotechnol 21(6):637–645
doi: 10.4014/jmb.1101.01036
Lobstein J et al (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11(1):753
doi: 10.1186/1475-2859-11-56
Röthlisberger D, Honegger A, Plückthun A (2005) Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J Mol Biol 347(4):773–789
doi: 10.1016/j.jmb.2005.01.053
Antibody molecules having specificity for human necrosis factor alpha, and use thereof. WO 01/94585A1
Haaland, P.D., Experimental design in biotechnology. Vol. 105. 1989: CRC press.
Box GE, Wilson KB (1951) On the experimental attainment of optimum conditions. J Roy Stat Soc: Ser B (Methodol) 13(1):1–38
Myers RH et al (2004) Response surface methodology: a retrospective and literature survey. J Qual Technol 36(1):53–77
doi: 10.1080/00224065.2004.11980252
Yee L, Blanch H (1992) Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Bio/Technology 10(12):1550–1556
Kram KE, Finkel SE (2015) Rich medium composition affects Escherichia coli survival, glycation, and mutation frequency during long-term batch culture. Appl Environ Microbiol 81(13):4442–4450
doi: 10.1128/AEM.00722-15
Marisch K et al (2013) Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Fact 12(1):58
doi: 10.1186/1475-2859-12-58
Winter J et al (2000) Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA. J Biotechnol 84(2):175–185
doi: 10.1016/S0168-1656(00)00356-4
Moore JT et al (1993) Overcoming inclusion body formation in a high-level expression system. Protein Expr Purif 4(2):160–163
doi: 10.1006/prep.1993.1022
Hoffmann F et al (2004) Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Technol 34(3–4):235–241
doi: 10.1016/j.enzmictec.2003.10.011
Iafolla MA et al (2008) Dark proteins: effect of inclusion body formation on quantification of protein expression. Proteins Structure Function Bioinformatics 72(4):1233–1242
doi: 10.1002/prot.22024
Siller E et al (2010) Slowing bacterial translation speed enhances eukaryotic protein folding efficiency. J Mol Biol 396(5):1310–1318
doi: 10.1016/j.jmb.2009.12.042
Yoon SK, Kang WK, Park TH (1994) Fed-batch operation of recombinant Escherichia coli containing trp promoter with controlled specific growth rate. Biotechnol Bioeng 43(10):995–999
doi: 10.1002/bit.260431013
Rezaie F et al (2017) Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode. J Appl Microbiol 123(1):134–144
doi: 10.1111/jam.13483
Jhamb K, Sahoo DK (2012) Production of soluble recombinant proteins in Escherichia coli: effects of process conditions and chaperone co-expression on cell growth and production of xylanase. Biores Technol 123:135–143
doi: 10.1016/j.biortech.2012.07.011
Chen Y et al (2003) DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Expr Purif 32(2):221–231
doi: 10.1016/S1046-5928(03)00233-X
Mazaheri S et al (2020) Improvement of Certolizumab Fab′ properties by PASylation technology. Sci Rep 10(1):1–13
doi: 10.1038/s41598-020-74549-0