A novel protein purification scheme based on salt inducible self-assembling peptides.
Controllable aggregating tag
Escherichia coli
Intein
Protein expression and purification
Salt-inducible self-assembling peptide
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
30 Oct 2023
30 Oct 2023
Historique:
received:
16
08
2023
accepted:
12
10
2023
medline:
31
10
2023
pubmed:
30
10
2023
entrez:
30
10
2023
Statut:
epublish
Résumé
Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.
Sections du résumé
BACKGROUND
BACKGROUND
Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release.
RESULTS
RESULTS
In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%.
CONCLUSION
CONCLUSIONS
Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.
Identifiants
pubmed: 37899435
doi: 10.1186/s12934-023-02229-5
pii: 10.1186/s12934-023-02229-5
pmc: PMC10614350
doi:
Substances chimiques
Peptides
0
Proteins
0
Sodium Chloride
451W47IQ8X
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
224Subventions
Organisme : National Key Research and Development Program of China
ID : 2018YFA0901000, 2022YFC2104800
Organisme : National Key Research and Development Program of China
ID : 2018YFA0901000, 2022YFC2104800
Organisme : Program for Guangdong Introducing Innovative and Entrepreneurial Teams
ID : 2019ZT08Y318
Informations de copyright
© 2023. The Author(s).
Références
Nat Protoc. 2006;1(6):2820-7
pubmed: 17406540
Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8414-9
pubmed: 15939888
Sci Rep. 2020 Jul 20;10(1):12002
pubmed: 32686735
Nucleic Acids Res. 2002 May 15;30(10):e43
pubmed: 12000848
Biotechnol J. 2017 Jun;12(6):
pubmed: 28296345
Elife. 2020 Apr 21;9:
pubmed: 32314955
Biotechnol Bioeng. 2020 Oct;117(10):2923-2932
pubmed: 32543719
Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7
pubmed: 22366317
Nat Methods. 2005 Sep;2(9):659-61
pubmed: 16074986
Sci Rep. 2014 Dec 01;4:7266
pubmed: 25434527
Front Bioeng Biotechnol. 2022 Jun 22;10:878838
pubmed: 35814018
Biotechnol Adv. 2020 Nov 15;44:107632
pubmed: 32971204
Science. 1979 Aug 10;205(4406):602-7
pubmed: 377496
Microb Cell Fact. 2011 Feb 15;10:9
pubmed: 21320350
Microb Cell Fact. 2011 Jun 02;10:42
pubmed: 21631955
Biochem Mol Biol Int. 1998 Jun;45(2):337-47
pubmed: 9678255
Nat Methods. 2009 May;6(5):343-5
pubmed: 19363495
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3334-8
pubmed: 7682699
Appl Microbiol Biotechnol. 2007 Nov;77(2):483-8
pubmed: 17768617
J Chromatogr A. 2011 Dec 9;1218(49):8826-35
pubmed: 21752387
Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12656-61
pubmed: 16129839
Nanoscale. 2021 Jun 3;13(21):9864-9872
pubmed: 34037034
Biotechnol Adv. 2020 Dec;45:107653
pubmed: 33157154
Enzyme Microb Technol. 2016 Nov;93-94:92-98
pubmed: 27702489
Protein Expr Purif. 2021 Dec;188:105974
pubmed: 34520839
Semin Cancer Biol. 2005 Oct;15(5):413-20
pubmed: 16061392
Chem Rev. 2020 Dec 23;120(24):13434-13460
pubmed: 33216525
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Apr 15;866(1-2):133-53
pubmed: 18294930
Nat Protoc. 2006;1(5):2326-33
pubmed: 17406475
Nat Methods. 2008 Feb;5(2):135-46
pubmed: 18235434
Acta Biomater. 2013 Nov;9(11):9075-85
pubmed: 23871942
Int J Biol Macromol. 2018 Oct 15;118(Pt B):2176-2184
pubmed: 30021136
Proc Natl Acad Sci U S A. 2017 Jun 27;114(26):E5138-E5147
pubmed: 28607052
Nanoscale. 2016 Apr 7;8(13):7127-36
pubmed: 26964879
Microb Cell Fact. 2016 Aug 05;15(1):136
pubmed: 27495238
Science. 2020 Oct 23;370(6515):426-431
pubmed: 32907861
Microb Cell Fact. 2005 Nov 11;4:32
pubmed: 16283936
Biotechnol J. 2015 Dec;10(12):1877-86
pubmed: 26556016
Trends Biotechnol. 2010 May;28(5):272-9
pubmed: 20359761
NPJ Regen Med. 2021 Feb 17;6(1):9
pubmed: 33597509
PLoS One. 2013;8(2):e56168
pubmed: 23409149
ACS Appl Mater Interfaces. 2014 Jun 11;6(11):8184-9
pubmed: 24821330
Anal Chem. 2022 May 10;94(18):6799-6808
pubmed: 35471023
Nat Biotechnol. 1999 Sep;17(9):889-92
pubmed: 10471931
Biotechniques. 2013 Apr;54(4):197-8, 200, 202, 204, 206
pubmed: 23581466