Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
© 2020 The Authors..
Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.
Medienart: |
E-Artikel |
---|
Erscheinungsjahr: |
2020 |
---|---|
Erschienen: |
2020 |
Enthalten in: |
Zur Gesamtaufnahme - volume:28 |
---|---|
Enthalten in: |
Biotechnology reports (Amsterdam, Netherlands) - 28(2020) vom: 03. Dez., Seite e00540 |
Sprache: |
Englisch |
---|
Beteiligte Personen: |
Tran, Dinh Thi Minh [VerfasserIn] |
---|
Links: |
---|
Anmerkungen: |
Date Revised 12.11.2020 published: Electronic-eCollection Citation Status PubMed-not-MEDLINE |
---|
doi: |
10.1016/j.btre.2020.e00540 |
---|
funding: |
|
---|---|
Förderinstitution / Projekttitel: |
|
PPN (Katalog-ID): |
NLM317311476 |
---|
LEADER | 01000naa a22002652 4500 | ||
---|---|---|---|
001 | NLM317311476 | ||
003 | DE-627 | ||
005 | 20231225163012.0 | ||
007 | cr uuu---uuuuu | ||
008 | 231225s2020 xx |||||o 00| ||eng c | ||
024 | 7 | |a 10.1016/j.btre.2020.e00540 |2 doi | |
028 | 5 | 2 | |a pubmed24n1057.xml |
035 | |a (DE-627)NLM317311476 | ||
035 | |a (NLM)33163371 | ||
040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
041 | |a eng | ||
100 | 1 | |a Tran, Dinh Thi Minh |e verfasserin |4 aut | |
245 | 1 | 0 | |a Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis |
264 | 1 | |c 2020 | |
336 | |a Text |b txt |2 rdacontent | ||
337 | |a ƒaComputermedien |b c |2 rdamedia | ||
338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
500 | |a Date Revised 12.11.2020 | ||
500 | |a published: Electronic-eCollection | ||
500 | |a Citation Status PubMed-not-MEDLINE | ||
520 | |a © 2020 The Authors. | ||
520 | |a Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis | ||
650 | 4 | |a Journal Article | |
650 | 4 | |a BgaB, β-galactosidase | |
650 | 4 | |a IPTG, isopropylthiogalactoside | |
650 | 4 | |a Inducer-free | |
650 | 4 | |a Integrative expression vector | |
650 | 4 | |a LB, Luria broth | |
650 | 4 | |a MCS, multiple cloning site | |
650 | 4 | |a MUG, methylumbelliferyl β-d-galactopyranoside | |
650 | 4 | |a Pgrac01 promoter | |
650 | 4 | |a Pgrac100 promoter | |
650 | 4 | |a Pgrac212 promoter | |
650 | 4 | |a pHT vector | |
700 | 1 | |a Phan, Trang Thi Phuong |e verfasserin |4 aut | |
700 | 1 | |a Doan, Thanh Thi Ngoc |e verfasserin |4 aut | |
700 | 1 | |a Tran, Thuoc Linh |e verfasserin |4 aut | |
700 | 1 | |a Schumann, Wolfgang |e verfasserin |4 aut | |
700 | 1 | |a Nguyen, Hoang Duc |e verfasserin |4 aut | |
773 | 0 | 8 | |i Enthalten in |t Biotechnology reports (Amsterdam, Netherlands) |d 2014 |g 28(2020) vom: 03. Dez., Seite e00540 |w (DE-627)NLM241422434 |x 2215-017X |7 nnns |
773 | 1 | 8 | |g volume:28 |g year:2020 |g day:03 |g month:12 |g pages:e00540 |
856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.btre.2020.e00540 |3 Volltext |
912 | |a GBV_USEFLAG_A | ||
912 | |a GBV_NLM | ||
951 | |a AR | ||
952 | |d 28 |j 2020 |b 03 |c 12 |h e00540 |