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.

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.

scholarly journals Cosecretion of Chaperones and Low-Molecular-Size Medium Additives Increases the Yield of Recombinant Disulfide-Bridged Proteins

2001 ◽  
Vol 67 (9) ◽  
pp. 3994-4000 ◽  
Author(s):  
Jörg Schäffner ◽  
Jeannette Winter ◽  
Rainer Rudolph ◽  
Elisabeth Schwarz
Keyword(s):  
Antibody Fragment ◽  
Molecular Size ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Positive Effects ◽  
Type Plasminogen Activator ◽  
Model Protein

ABSTRACT Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive l-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 Ml-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm ofE. coli.

scholarly journals Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis: Comparison of Sec and Tat Pathway Export Capabilities

2006 ◽  
Vol 72 (7) ◽  
pp. 4589-4595 ◽  
Author(s):  
Melloney J. Dröge ◽  
Ykelien L. Boersma ◽  
Peter G. Braun ◽  
Robbert Jan Buining ◽  
Mattijs K. Julsing ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phage Display ◽  
Signal Peptide ◽  
Signal Peptides ◽  
Coat Proteins ◽  
Phage Display Technology ◽  
E Coli ◽  
Tat Pathway ◽  
Dependent Signal ◽  
Display Technology

ABSTRACT Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

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