The Tat pathway as a biotechnological tool for the expression and export of heterologous proteins inEscherichia coli

2015 ◽  
Vol 3 (6) ◽  
pp. 387-396 ◽  
Author(s):  
Kelly L Walker ◽  
Alexander S Jones ◽  
Colin Robinson
Keyword(s):  
Inescherichia Coli ◽  
Heterologous Proteins ◽  
Tat Pathway

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

Constitutive versus thermoinducible expression of heterologous proteins inEscherichia coli based on strong PR,PL promoters from phage lambda

2003 ◽  
Vol 83 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Viktor Menart ◽  
Simona Jev?evar ◽  
Mateja Vilar ◽  
Andreja Trobi? ◽  
Aleksander Pavko
Keyword(s):  
Inescherichia Coli ◽  
Heterologous Proteins ◽  
Phage Lambda

scholarly journals Requirement for phospholipids of the translocation of the trimethylamineN-oxide reductase through the Tat pathway inEscherichia coli

FEBS Letters ◽  
1999 ◽  
Vol 463 (3) ◽  
pp. 331-335 ◽  
Author(s):  
Nathalia I. Mikhaleva ◽  
Claire-Lise Santini ◽  
Gérard Giordano ◽  
Marina A. Nesmeyanova ◽  
Long-Fei Wu
Keyword(s):  
Inescherichia Coli ◽  
Tat Pathway

scholarly journals Engineering a super-secreting strain of Escherichia coli by directed co-evolution of the multiprotein Tat translocation machinery

2021 ◽  
Author(s):  
May N. Taw ◽  
Mingji Li ◽  
Daniel Kim ◽  
Mark A. Rocco ◽  
Dujduan Waraho-Zhmayev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Genetic Manipulation ◽  
Value Added ◽  
Heterologous Proteins ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Transit Times ◽  
Tat Pathway ◽  
Folded Proteins

AbstractEscherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed co-evolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three super-secreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than that observed for wildtype TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of highly efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

scholarly journals The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

2008 ◽  
Vol 74 (24) ◽  
pp. 7507-7513 ◽  
Author(s):  
Marc A. B. Kolkman ◽  
René van der Ploeg ◽  
Michael Bertels ◽  
Maurits van Dijk ◽  
Joop van der Laan ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Heterologous Proteins ◽  
Independent Manner ◽  
Sec Pathway ◽  
Tat Pathway ◽  
Cargo Proteins ◽  
Dependent Protein ◽  
Application Potential ◽  
Tat System

ABSTRACT Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.

scholarly journals Engineering of the Bacillus subtilis PhoD signal peptide to direct high-level, Tat-specific export of a single-chain antibody fragment to the periplasm in Eschericha coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background : Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results : We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions : The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

Versatile signal peptide ofFlavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins inEscherichia coli

2016 ◽  
Vol 32 (4) ◽  
pp. 848-854 ◽  
Author(s):  
Dong Gyun Kang ◽  
Jeong Hyun Seo ◽  
Byung Hoon Jo ◽  
Chang Sup Kim ◽  
Suk Soon Choi ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Organophosphorus Hydrolase ◽  
Inescherichia Coli ◽  
Heterologous Proteins
Sign in / Sign up

Export Citation Format

Share Document