Abstract
Expression of recombinant protein that possess disulfide bonds especially Fab antibody fragment in periplasm of E. coli is the most favorable platform. But formation of inclusion bodies and inefficient translocation of desired protein to the periplasm is considerable obstacle threatening the advantages of E. coli expression system. Co-expression with molecular chaperones is remarkable consideration to solve this problem. In this study we evaluated the effect of co-expression of molecular chaperones and optimization condition on efficient periplasmic translocation of Fab antibody fragment. Plasmid pKJE7 was used for co-expression of DnaK/DnaJ/GrpE in BL21 cells and SHuffle strain (constantly expressing DsbC chaperon) were used for evaluation of chaperones effects. Periplasmic fraction was prepared by osmotic shock method and ELISA test were used for activity measurement. The Fab antibody purified by IMAC chromatography. The results indicated that co-expression of anti-TNF-α Fab with DnaK/DnaJ/GrpE in BL21 cells had a marked effect on the yield of periplasmic fraction but DsbC had no effect on periplasmic translocation. The optimal culture condition was found 0.1 mM IPTG concentration and 25 °C temperatures. SDS-PAGE analysis indicated that solubility increased to 42% of total Fab in Bl21 co-expressing DnaKJE. This study reports that co-expression with DnaK/DnaJ/GrpE markedly increases soluble expression of Fab antibody fragment in optimized condition.
Similar content being viewed by others
References
Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399
Cannon-Carlson S, Tang J (1997) Modification of the Laemmli sodium dodecyl sulfate–polyacrylamide gel electrophoresis procedure to eliminate artifacts on reducing and nonreducing gels. Anal Biochem 246:146–148
Chen J, Song J-l, Zhang S, Wang Y, Cui D-F, Wang C-C (1999) Chaperone activity of DsbC. J Biol Chem 274:19601–19605
Choi J, Lee S (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635
Chung KT, Lee TH, Kang GS (2003) Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone. Biotechnol Bioprocess Eng 8:192–198
Dariushnejad H FS, Zarghami N, ahdi Khosroshahi S, Rahbarnia l (2017) Dsbc chaperone mediated soluble expression of human tNF-α in E. coli. Minerva Biotechnol. doi:10.23736/S1120-4826.17.02299-6
Davidson VL, Sun D (2002) [12]-lysozyme-osmotic shock methods for localization of periplasmic redox proteins in bacteria. Methods Enzymol 353:121–130
Duilio A, Tutino ML, Marino G (2004) Recombinant protein production in Antarctic Gram-negative bacteria. Methods Mol Biol 267:225–237
Dumitru GL, Groemping Y, Klostermeier D, Restle T, Deuerling E, Reinstein J (2004) DafA cycles between the DnaK chaperone system and translational machinery. J Mol Biol 339:1179–1189
Ellis M, Patel P, Edon M, Ramage W, Dickinson R, Humphreys D (2017) Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab’fragments. Biotechnol Prog 33:212–220
Frenzel A, Hust M, Schirrmann T (2013) Expression of recombinant antibodies. Front Immunol 4:217–237
Fu X-Y (2010) Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium. Appl Microbiol Biotechnol 88:75–86
Gupta SK, Shukla P (2016) Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 36:1089–1098
Gupta SK, Shukla P (2017) Microbial platform technology for recombinant antibody fragment production: a review. Crit Rev Biotechnol 43:31–42
Hsu CC, Thomas OR, Overton TW (2016) Periplasmic expression in and release of Fab fragments from Escherichia coli using stress minimization. J Chem Technol Biotechnol 91:815–822
Hu X, O’Hara L, White S, Magner E, Kane M, Wall JG (2007) Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support. Protein Expr Purif 52:194–201
Kang H-J, Kim H-J, Jung M-S, Han J-K, Cha S-H (2017) Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule. Immunol Lett 184:34–42
Kurt N, Rajagopalan S, Cavagnero S (2006) Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain. J Mol Biol 355:809–820
Lee HC, Bernstein HD (2002) Trigger factor retards protein export in Escherichia coli. J Biol Chem 277:43527–43535
Levy R, Weiss R, Chen G, Iverson BL, Georgiou G (2001) Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones. Protein Expr Purif 23:338–347
Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial Cell Factor 11:753
Perezperez J, Martinezcaja C, Barbero JL, Gutierrez J (1995) DnaK/DnaJ supplementation improves the periplasmic production of human granulocyte-colony stimulating factor in Escherichia coli. Biochem Biophys Res Commun 210:524–529
Rodriguez C, Nam DH, Kruchowy E, Ge X (2017) Efficient antibody assembly in E. coli periplasm by disulfide bond folding factor co-expression and culture optimization. Appl Biochem Biotechnol 183:520–529
Schlieker C, Bukau B, Mogk A (2002) Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology. J Biotechnol 96:13–21
Sonoda H, Kumada Y, Katsuda T, Yamaji H (2011) Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli. J Biosci Bioeng 111:465–470
Sørensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factor 4:1
Taylor T, Denson J-P, Esposito D (2017) Optimizing expression and solubility of proteins in E. coli using modified media and induction parameters. Methods Mol Biol 1586:65–82
Wan EW-M, Baneyx F (1998) TolAIII co-overexpression facilitates the recovery of periplasmic recombinant proteins into the growth medium of Escherichia coli. Protein Expr Purif 14:13–22
Ying B-W, Taguchi H, Ueda H, Ueda T (2004) Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system. Biochem Biophys Res Commun 320:1359–1364
Acknowledgements
This study was funded by Drug Applied Research Center (Grant No. 94/54), Tabriz University of Medical Science, Tabriz, Iran. The results presented in this work are based on data set of PhD thesis, recorded in Tabriz University of Medical Sciences.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Dariushnejad, H., Farajnia, S., Zarghami, N. et al. Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway. Int J Pept Res Ther 25, 67–74 (2019). https://doi.org/10.1007/s10989-017-9637-x
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10989-017-9637-x