Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene.
CRISPR/Cas9
RNA-seq analysis
caspase 8-associated protein 2
cyclin-dependent kinase inhibitor 2A
recombinant protein
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
22
02
2020
revised:
29
07
2020
accepted:
03
09
2020
pubmed:
11
9
2020
medline:
26
11
2021
entrez:
10
9
2020
Statut:
ppublish
Résumé
Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.
Identifiants
pubmed: 32910455
doi: 10.1002/bit.27561
pmc: PMC9621094
mid: NIHMS1838465
doi:
Substances chimiques
Apoptosis Regulatory Proteins
0
CASP8AP2 protein, human
0
CDKN2A protein, human
0
Calcium-Binding Proteins
0
Cyclin-Dependent Kinase Inhibitor p16
0
Recombinant Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Intramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
186-198Subventions
Organisme : Intramural NIH HHS
ID : Z01 DK070010
Pays : United States
Organisme : NIDDK NIH HHS
Pays : United States
Informations de copyright
© 2020 Wiley Periodicals LLC.
Références
Biotechnol Bioeng. 2015 Aug;112(8):1632-43
pubmed: 25676429
RNA Biol. 2013 Feb;10(2):287-300
pubmed: 23353574
Eur J Pharm Biopharm. 2011 Jun;78(2):184-8
pubmed: 21256214
Biotechnol Adv. 2015 Dec;33(8):1878-96
pubmed: 26523782
Biotechnol Bioeng. 2016 Nov;113(11):2403-15
pubmed: 27215166
Cytotechnology. 2010 Aug;62(4):301-6
pubmed: 20697810
Biotechnol Bioeng. 2002 Sep 20;79(6):619-27
pubmed: 12209809
Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6289-93
pubmed: 7603984
Curr Opin Cell Biol. 2000 Dec;12(6):676-84
pubmed: 11063931
Appl Microbiol Biotechnol. 2003 Dec;63(2):164-73
pubmed: 12856162
Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14808-12
pubmed: 17003125
Curr Opin Microbiol. 2004 Dec;7(6):631-7
pubmed: 15556036
Oncogene. 2008 Aug 7;27(34):4644-56
pubmed: 18408764
Biotechnol Prog. 1998 Sep-Oct;14(5):705-13
pubmed: 9758659
Methods Mol Biol. 2018;1850:209-219
pubmed: 30242689
Lab Anim (NY). 2019 Oct;48(10):282-284
pubmed: 31477900
Biotechnol J. 2018 Feb;13(2):
pubmed: 28987030
Bio Protoc. 2018 Sep 5;8(17):
pubmed: 30263905
Biotechnol Bioeng. 2008 Dec 15;101(6):1234-44
pubmed: 18980186
Biotechnol Adv. 2010 May-Jun;28(3):385-94
pubmed: 20156545
PLoS One. 2014 Sep 19;9(9):e108032
pubmed: 25238250
Biotechnol Bioeng. 2004 Mar 30;85(7):741-9
pubmed: 14991652
Oncogene. 2010 Feb 11;29(6):802-10
pubmed: 19915611
Nature. 1999 Apr 29;398(6730):777-85
pubmed: 10235259
Cytotechnology. 2001 Jul;36(1-3):71-83
pubmed: 19003317
Mol Cell. 2009 Oct 23;36(2):267-78
pubmed: 19854135
Mol Cancer. 2012 Jan 04;11:1
pubmed: 22216762
Sci Rep. 2019 Jun 18;9(1):8689
pubmed: 31213643
Oncogene. 2005 Jan 20;24(4):688-96
pubmed: 15592525
Biotechnol Bioeng. 2004 Jan 20;85(2):177-84
pubmed: 14705000
Mol Cell Biol. 2009 Sep;29(17):4729-41
pubmed: 19546234
Biotechnol Bioeng. 2015 Jan;112(1):141-55
pubmed: 25042542
Biotechnol Bioeng. 2008 Aug 15;100(6):1178-92
pubmed: 18553404
Methods Mol Biol. 2017;1603:71-85
pubmed: 28493124
Biotechnol Bioeng. 2010 Feb 1;105(2):330-40
pubmed: 19777580
Cytotechnology. 1999 Jul;30(1-3):85-93
pubmed: 19003358
Oncogene. 2012 Feb 2;31(5):573-82
pubmed: 21725362
Biotechnol Prog. 2003 Jan-Feb;19(1):224-8
pubmed: 12573029
Biotechnol Bioeng. 1997 Sep 20;55(6):927-39
pubmed: 18636602
Biotechnol Bioeng. 2002 Mar 5;77(5):528-37
pubmed: 11788951