The twin-arginine translocation (Tat) system.

In this Quick guide, Palmer and Berks introduce the twin-arginine translocation (Tat) systems. Tats are found in a variety of microbes and microbe-derived organelles, and are known to translocate folded substrate proteins across biological membranes.

A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

Highly efficient export of a disulfide-bonded protein to the periplasm and medium by the Tat pathway using CyDisCo in Escherichia coli.

High-value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well-studied TorA signal peptide that is Tat-specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond-containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level.

Analysis of the Brucella suis Twin Arginine Translocation System and Its Substrates Shows That It Is Essential for Viability.

Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gram-negative bacteria, the Tat system of the zoonotic pathogen Brucella spp. is encoded by a three-gene operon, tatABC. Our attempts, using several different strategies, to create a Brucella suis strain 1330 tat mutant were all unsuccessful. This suggested that, for B. suis, Tat is essential, in contrast to a recent report for Brucella melitensis. This was supported by our findings that two molecules that inhibit the Pseudomonas aeruginosa Tat system also inhibit B. suis, B. melitensis, and Brucella abortus growth in vitro. In a bioinformatic screen of the B. suis 1330 proteome, we identified 28 proteins with putative Tat signal sequences. We used a heterologous reporter assay based on export of the Tat-dependent amidase AmiA by using the Tat signal sequences from the Brucella proteins to confirm that 20 of the 28 candidates can engage the Tat pathway.

Envelope Stress Activates Expression of the Twin Arginine Translocation (Tat) System in Salmonella.

The twin arginine translocation system (Tat) is a protein export system that is conserved in bacteria, archaea, and plants. In Gram-negative bacteria, it is required for the export of folded proteins from the cytoplasm to the periplasm. In Salmonella, there are 30 proteins that are predicted substrates of Tat, and among these are enzymes required for anaerobic respiration and peptidoglycan remodeling. We have demonstrated that some conditions that induce bacterial envelope stress activate expression of a ΔtatABC-lacZ fusion in Salmonella enterica serovar Typhimurium. Particularly, the addition of bile salts to the growth medium causes a 3-fold induction of a ΔtatABC-lacZ reporter fusion. Our data demonstrate that this induction is mediated via the phage shock protein (Psp) stress response system protein PspA. Further, we show that deletion of tatABC increases the induction of tatABC expression in bile salts. Indeed, the data suggest significant interaction between PspA and the Tat system in the regulatory response to bile salts. Although we have not identified the precise mechanism of Psp regulation of tatABC, our work shows that PspA is involved in the activation of tatABC expression by bile salts and adds another layer of complexity to the Salmonella response to envelope stress. IMPORTANCE Salmonella species cause an array of diseases in a variety of hosts. This research is significant in showing induction of the Tat system as a defense against periplasmic stress. Understanding the underlying mechanism of this regulation broadens our understanding of the Salmonella stress response, which is critical to the ability of the organism to cause infection.

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli.

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and-together with laterally-aligned tilted amphipathic helices-generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.

Membrane translocation of folded proteins.

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.

The Rcs System Contributes to the Motility Defects of the Twin-Arginine Translocation System Mutant of Extraintestinal Pathogenic Escherichia coli.

Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δtat, the ΔmdoD, and the ΔamiAΔamiC strains, which restored the motility of ΔmdoD and partially restored the motility of Δtat and ΔamiAΔamiC. The flagella were also observed in all of the ΔtatΔrcsDB, ΔmdoDΔrcsDB, and ΔamiAΔamiCΔrcsDB strains, but not in the Δtat, ΔmdoD, and ΔamiAΔamiC strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the tat mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the tat mutant of ExPEC. IMPORTANCE The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the tat mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.

Tat-SynGAP improves angiogenesis and post-stroke recovery by inhibiting MST1/JNK signaling.

Small G protein Ras induces the activation of apoptosis-related molecule mammalian Ste20-like kinase1 (MST1)/JNK signal pathway, which is involved in the regulation of tissue damage under pathological conditions such as ischemic stroke. Our previous study indicated that GTPase-activating protein for Ras (SynGAP), a negative regulator of Ras, could bind with postsynaptic density protein-93 (PSD-93) and Tat-SynGAP (670-685aa) small peptide to exhibit neuroprotective role. Here, we report that Tat-SynGAP (670-685aa) reduced cerebral edema at acute cerebral ischemia/reperfusion (I/R), improved integrity of blood-brain barrier, and decreased cortical and striatum neuronal injury. Mechanistically, Tat-SynGAP (670-685aa) not only inhibited the phosphorylation of MST1 and JNK and the cleavage of caspase-3, but also facilitated the expression of angiogenesis related molecules VEGF and Ang-1. In conclusion, Tat-SynGAP (670-685aa) reduces neuronal apoptosis and cerebral infarction volume and maintains vascular stability and blood-brain barrier integrity by inhibiting MST1/JNK signaling pathway.

Engineering a Supersecreting Strain of Escherichia coli by Directed Coevolution of the Multiprotein Tat Translocation Machinery.

Escherichia 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 coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type 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 more 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.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation.

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 µM), and this monomer binds reversibly to spTorA (KD ≈ 1 µM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Double trouble: Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment.

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

The Tat system and its dependent cell division proteins are critical for virulence of extra-intestinal pathogenic Escherichia coli.

The twin-arginine translocation (Tat) system is involved in a variety of important bacterial physiological processes. Conserved among bacteria and crucial for virulence, the Tat system is deemed as a promising anti-microbial drug target. However, the mechanism of how the Tat system functions in bacterial pathogenesis has not been fully understood. In this study, we showed that the Tat system was critical for the virulence of an extra-intestinal pathogenic E. coli (ExPEC) strain PCN033. A total of 20 Tat-related mutant strains were constructed, and competitive infection assays were performed to evaluate the relative virulence of these mutants. The results demonstrated that several Tat substrate mutants, including the ΔsufI, ΔamiAΔamiC double mutant as well as each single mutant, ΔyahJ, ΔcueO, and ΔnapG, were significantly outcompeted by the WT strain, among which the ΔsufI and ΔamiAΔamiC strains showed the lowest competitive index (CI) value. Results of individual mouse infection assay, in vitro cell adhesion assay, whole blood bactericidal assay, and serum bactericidal assay further confirmed the virulence attenuation phenotype of the ΔsufI and ΔamiAΔamiC strains. Moreover, the two mutants displayed chained morphology in the log phase resembling the Δtat and were defective in stress response. Our results suggest that the Tat system and its dependent cell division proteins SufI, AmiA, and AmiC play critical roles during ExPEC pathogenesis.

The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis.

Brucella, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H2O2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria.

The biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA. Here we show that the nuclearly encoded TatA from pea has dual targeting properties, i.e., it can be imported into both, chloroplasts and mitochondria. Dual targeting of TatA was observed with in organello experiments employing chloroplasts and mitochondria isolated from pea as well as after transient expression of suitable reporter constructs in leaf tissue from pea and Nicotiana benthamiana. The extent of transport of these constructs into mitochondria of transiently transformed leaf cells was relatively low, causing a demand for highly sensitive methods to be detected, like the sasplitGFP approach. Yet, the dual import of TatA into mitochondria and chloroplasts observed here points to a common mechanism of Tat transport for folded proteins within both endosymbiotic organelles in plants.

Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting.

In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.

Targeting of proteins to the twin-arginine translocation pathway.

The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N-terminal signal sequence containing a highly conserved twin-arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.

Twin-Arginine Translocation System Is Involved in Citrobacter rodentium Fitness in the Intestinal Tract.

The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

OBJECTIVES: The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. RESULTS: In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5 kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. CONCLUSIONS: In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.