Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of Escherichia coli-a rate limiting step in the induction of recombinant protein expression.
Escherichia coli
Active transport
IPTG
Simple diffusion
lacY
Journal
Journal of industrial microbiology & biotechnology
ISSN: 1476-5535
Titre abrégé: J Ind Microbiol Biotechnol
Pays: Germany
ID NLM: 9705544
Informations de publication
Date de publication:
17 Feb 2023
17 Feb 2023
Historique:
received:
20
05
2023
accepted:
16
10
2023
medline:
13
11
2023
pubmed:
18
10
2023
entrez:
18
10
2023
Statut:
ppublish
Résumé
Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts. A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts.
Identifiants
pubmed: 37849239
pii: 7320299
doi: 10.1093/jimb/kuad034
pmc: PMC10639102
pii:
doi:
Substances chimiques
beta-galactoside
0
Isopropyl Thiogalactoside
367-93-1
Galactosides
0
Recombinant Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Fundação de Amparo à Pesquisa do Estado de São Paulo
ID : 2018/24926-3
Organisme : Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
ID : PVE-X88881.339140/2019-01
Informations de copyright
© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.
Références
Curr Biol. 2015 Feb 2;25(3):385-391
pubmed: 25544609
Biochemistry. 1991 Feb 5;30(5):1284-90
pubmed: 1991109
J Bacteriol. 1959 Nov;78:613-23
pubmed: 13811042
Biochim Biophys Acta. 1973 Dec 13;330(2):196-205
pubmed: 4591126
J Theor Biol. 2009 Jan 7;256(1):14-28
pubmed: 18848570
Proc Natl Acad Sci U S A. 1984 Mar;81(6):1629-33
pubmed: 6324209
Ann Inst Pasteur (Paris). 1956 Dec;91(6):829-57
pubmed: 13395026
Adv Biochem Eng Biotechnol. 2004;89:73-92
pubmed: 15217156
J Mol Biol. 1986 May 5;189(1):113-30
pubmed: 3537305
Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8440-5
pubmed: 24872451
J Chem Theory Comput. 2006 Sep;2(5):1416-28
pubmed: 26626849
Physiol Rev. 1990 Oct;70(4):1135-76
pubmed: 2217557
Mol Biosyst. 2015 Sep;11(9):2579-87
pubmed: 26223179
Proc Natl Acad Sci U S A. 1957 Jul 15;43(7):553-66
pubmed: 16590055
Nature. 2011 Jul 20;475(7356):308-15
pubmed: 21776076
Eur J Biochem. 1984 Feb 1;138(3):497-508
pubmed: 6363073
Biochemistry. 1980 Dec 9;19(25):5692-702
pubmed: 7006690
Sci Rep. 2018 Apr 3;8(1):5576
pubmed: 29615663
Trends Microbiol. 2018 Oct;26(10):815-832
pubmed: 29843923
Science. 2010 Jul 30;329(5991):533-8
pubmed: 20671182
J Gen Microbiol. 1958 Dec;19(3):592-606
pubmed: 13611202
J Mol Biol. 1961 Jun;3:318-56
pubmed: 13718526
Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4083-8
pubmed: 15016920
Methods Mol Biol. 2014;1091:17-32
pubmed: 24203322
Metab Eng. 2017 Jul;42:145-156
pubmed: 28645641
Biophys J. 2001 Oct;81(4):2010-9
pubmed: 11566774
Nature. 2004 Feb 19;427(6976):737-40
pubmed: 14973486
Microbiol Mol Biol Rev. 1998 Mar;62(1):110-29
pubmed: 9529889
C R Hebd Seances Acad Sci. 1964 Mar 16;258:3125-8
pubmed: 14143651
Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12173-8
pubmed: 21730194
J Physiol. 1952 Sep;118(1):23-39
pubmed: 13000688
Protein Expr Purif. 2005 Jan;39(1):54-60
pubmed: 15596360
J Biol Chem. 1966 May 25;241(10):2200-11
pubmed: 5330114
Biochim Biophys Acta. 1975 Aug 20;401(2):285-98
pubmed: 1098694
Elife. 2019 Aug 28;8:
pubmed: 31456563
J Chem Phys. 2019 Mar 14;150(10):104705
pubmed: 30876365
Nat Commun. 2021 Mar 30;12(1):1975
pubmed: 33785742
Biotechnol Bioeng. 2016 Apr;113(4):772-82
pubmed: 26416399
Eur J Biochem. 2000 Jan;267(2):527-34
pubmed: 10632722
J Cell Biol. 2003 May 12;161(3):471-6
pubmed: 12743100
Biophys J. 2003 May;84(5):2841-51
pubmed: 12719218
Biophys J. 2004 Mar;86(3):1282-92
pubmed: 14990461
Biochem Biophys Res Commun. 1980 Sep 30;96(2):656-61
pubmed: 6775629
J Biotechnol. 2012 Jan;157(1):82-8
pubmed: 22079752
EMBO J. 1990 Apr;9(4):973-9
pubmed: 2182324
J Bacteriol. 2003 Mar;185(5):1485-91
pubmed: 12591863
Mol Pharm. 2018 Apr 2;15(4):1488-1494
pubmed: 29462563
Bacteriol Rev. 1976 Sep;40(3):527-51
pubmed: 186018
Science. 2008 Oct 17;322(5900):442-6
pubmed: 18927393
Data Brief. 2014 Aug 22;1:7-11
pubmed: 26217677
Biophys J. 1996 Jan;70(1):339-48
pubmed: 8770210
Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15147-51
pubmed: 21896727
Biochim Biophys Acta. 1960 May 6;40:70-84
pubmed: 14408566
J Biotechnol. 2012 Feb 10;157(3):391-8
pubmed: 22202176
Proc Natl Acad Sci U S A. 1970 Jul;66(3):773-9
pubmed: 4913210
Nat Biotechnol. 2009 Oct;27(10):946-50
pubmed: 19801975
J Mol Biol. 1965 May;12:88-118
pubmed: 14343300
J Gen Physiol. 2019 Jul 1;151(7):878-886
pubmed: 31147449
Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15912-7
pubmed: 25349411
PLoS Comput Biol. 2012;8(12):e1002811
pubmed: 23271961
Nature. 2005 Jul 28;436(7050):588-92
pubmed: 16049495
Front Mol Biosci. 2018 Nov 13;5:93
pubmed: 30483513
Biophys J. 1997 Jan;72(1):223-37
pubmed: 8994607
Cell Syst. 2018 Apr 25;6(4):456-469.e10
pubmed: 29574055
Microb Cell Fact. 2020 Apr 6;19(1):83
pubmed: 32252765
PLoS One. 2011;6(7):e23126
pubmed: 21829590
Antimicrob Agents Chemother. 2017 Sep 22;61(10):
pubmed: 28717042