Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process.
Bacterial protein production
Inclusion bodies
Mild solubilization
Refolding
Single chain variable fragment
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
14 Jan 2019
14 Jan 2019
Historique:
received:
06
09
2018
accepted:
04
01
2019
entrez:
16
1
2019
pubmed:
16
1
2019
medline:
2
2
2019
Statut:
epublish
Résumé
The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein. It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer. Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.
Sections du résumé
BACKGROUND
BACKGROUND
The production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein.
RESULTS
RESULTS
It was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation-refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer.
CONCLUSIONS
CONCLUSIONS
Maltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.
Identifiants
pubmed: 30642336
doi: 10.1186/s12934-019-1053-9
pii: 10.1186/s12934-019-1053-9
pmc: PMC6330739
doi:
Substances chimiques
Maltose-Binding Proteins
0
Recombinant Proteins
0
Single-Chain Antibodies
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
5Subventions
Organisme : South Asian University
ID : Start-up fund
Références
Biotechnol Prog. 1998 Sep-Oct;14(5):722-8
pubmed: 9758661
Protein Sci. 1999 Aug;8(8):1668-74
pubmed: 10452611
Biotechnol J. 2012 May;7(5):620-34
pubmed: 22442034
Curr Pharm Biotechnol. 2010 Apr;11(3):309-12
pubmed: 20210737
Clin Diagn Lab Immunol. 2003 Jul;10(4):587-95
pubmed: 12853390
Methods Enzymol. 2000;326:312-21
pubmed: 11036650
Front Microbiol. 2014 Feb 14;5:56
pubmed: 24592259
Protein Expr Purif. 2012 Jan;81(1):75-82
pubmed: 21964443
Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13029-33
pubmed: 10557267
Front Microbiol. 2014 Sep 15;5:486
pubmed: 25309524
Microb Cell Fact. 2016 Jun 08;15:100
pubmed: 27277580
EMBO J. 2009 Oct 21;28(20):3269-76
pubmed: 19713934
FASEB J. 1996 Jan;10(1):49-56
pubmed: 8566547
Protein Expr Purif. 2011 May;77(1):68-74
pubmed: 21195184
J Biosci Bioeng. 2005 Apr;99(4):303-10
pubmed: 16233795
J Bacteriol. 2005 May;187(10):3599-601
pubmed: 15866952
Microb Cell Fact. 2015 Mar 25;14:41
pubmed: 25889252
Protein Expr Purif. 2005 Jun;41(2):341-8
pubmed: 15866720
Biomolecules. 2014 Feb 20;4(1):235-51
pubmed: 24970214
Bioinformatics. 2013 Jul 15;29(14):1750-7
pubmed: 23681122
Microb Cell Fact. 2010 Sep 10;9:66
pubmed: 20831775
Methods Enzymol. 2009;463:239-58
pubmed: 19892176
Biochem Biophys Res Commun. 2007 Dec 21;364(3):639-44
pubmed: 17964542
Biotechnol Prog. 2005 Mar-Apr;21(2):632-9
pubmed: 15801811
Nat Protoc. 2006;1(6):2527-35
pubmed: 17406506
World J Microbiol Biotechnol. 2018 Oct 19;34(11):158
pubmed: 30341583
PLoS One. 2018 Jul 18;13(7):e0201060
pubmed: 30021008
Methods Enzymol. 2009;463:259-82
pubmed: 19892177
Microb Cell Fact. 2014 Oct 02;13:141
pubmed: 25270715
Appl Microbiol Biotechnol. 2013 Aug;97(15):6779-91
pubmed: 23160981
Front Microbiol. 2014 Feb 19;5:63
pubmed: 24600443
Biotechnol Adv. 2009 May-Jun;27(3):297-306
pubmed: 19500547
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul 15;877(22):2045-51
pubmed: 19523887
Curr Protoc Protein Sci. 2001 May;Appendix 3:Appendix 3A
pubmed: 18429069
Sci Pharm. 2016 Feb 14;84(1):141-52
pubmed: 27110505
Gene. 1988 Jul 15;67(1):31-40
pubmed: 3047011
Curr Opin Biotechnol. 2001 Apr;12(2):202-7
pubmed: 11287238
Braz J Med Biol Res. 2014 Jul;47(7):540-7
pubmed: 24919171
Methods Mol Biol. 2014;1060:171-84
pubmed: 24037842
Appl Microbiol Biotechnol. 2006 Sep;72(2):211-22
pubmed: 16791589
Protein Sci. 1997 Feb;6(2):399-406
pubmed: 9041642
J Mol Biol. 2001 Sep 7;312(1):79-93
pubmed: 11545587
Protein Sci. 2002 Apr;11(4):739-56
pubmed: 11910019
Methods Mol Biol. 2015;1258:283-91
pubmed: 25447870
J Immunol Methods. 1998 Oct 1;219(1-2):119-29
pubmed: 9831393