From 2007.igem.org

• <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=235035">J Bacteriol. 1977 February; 129(2): 967–972</a>.
  • Isolation of an E. coli mutant deficient in thioredoxin reductase. (E. coli KK1048) </li>
  • A mutant of E. coli defective in thioredoxin reductase has been isolated and partially characterized. </li>
  • This mutant has no detectable thioredoxin reductase activity in vitro and yet it exhibits no in vivo defect in reduction of ribonucleotides. Evidence is presented that indicates that, in cells permeabilized via ether treatment, ribonucleoside diphosphate reduction can utilize glutathione as an alternate reducing system. </li>
• <a href="http://www.sciencemag.org/cgi/content/abstract/262/5140/1744">Science, Vol 262, Issue 5140, 1744-1747, 1993</a>.
  • Mutations that allow disulfide bond formation in the cytoplasm of E. coli.
  • Disulfide bonds are rarely found in cytoplasmic proteins.
  • Mutations were selected for in E. coli that allow disulfide bond formation in the cytoplasm. In the presence of these mutations, export-defective versions of alkaline phosphatase and mouse urokinase were able to fold into their enzymatically active conformations in the cytoplasm because their disulfide bonds were formed.
  • The mutations were mapped to the gene for thioredoxin reductase and diminish or eliminate the activity of this enzyme. Thioredoxin itself was found to be unnecessary for this disulfide bond formation. Thioredoxin reductase, but not thioredoxin, is thus implicated in keeping cysteines reduced in cytoplasmic proteins. 
• <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=40909">Proc Natl Acad Sci U S A. 1995 October 10; 92(21): 9895–9899.</a>
  • Disulfide bond formation is catalyzed in the periplasm of E. coli.
  • This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA.
  • Here we present direct evidence of an interaction between DsbA and DsbB.
• <a href="http://jb.asm.org/cgi/content/abstract/179/21/6602">J. Bacteriol., Nov 1997, 6602-6608, Vol 179, No. 21.</a>
  • Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.
  • The E. coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase.
  • For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form.
  • reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway.
  • The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds.
• <a href="http://www.nature.com/emboj/journal/v17/n19/abs/7591249a.html">The EMBO Journal (1998) 17, 5543–5550.</a>
  • Disulfide bond formation in the E. coli cytoplasm: an in vivo role reversal for the thioredoxins.
  • Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in E. coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway.
  • However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation.
  • Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth.
  • Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.
    • The formation of structural disulfide bonds in E. coli appears to be strictly segregated according to subcellular
      compartment.
      • In the periplasm, disulfide bonds are actively formed in many proteins by the Dsb system.
      • In the cytoplasm, the only disulfide bonds known to be present in proteins are formed in enzymes like ribonucleotide reductase during their catalytic cycles or in the oxidative response transcription factor OxyR during its regulatory cycle.
    • The reduced forms of these proteins are regenerated via the action of 
      <a href="http://igem.ym.edu.tw/private_2007/index.php/Image:Disulfide_reducing.jpg">the thioredoxin and glutathione/glutaredoxin pathways</a>.
    • In the thioredoxin pathway, thioredoxin reductase (the product of the trxB gene) uses the reducing potential of
      NADPH to maintain thioredoxin 1 (the product of the trxA gene) in the reduced state, so that thioredoxin 1 in
      turn can reduce substrate proteins such as ribonucleotide reductase.
    • The glutathione/glutaredoxin system also uses the reducing potential of NADPH in this case to reduce glutathione via the enzyme glutathione oxidoreductase (the product of the gor gene). Glutathione is then able to reduce the three glutaredoxins (glutaredoxin 1, 2 and 3). Only glutaredoxin 1 is able to reduce ribonucleotide reductase efficiently in vitro, whereas glutaredoxin 3 has a modest ability to reduce this enzyme. Although glutaredoxins 2 and 3 are less efficient at reducing protein disulfides, they are active in reducing mixed disulfides of glutathione.
    • Such mixed disulfides are generated and must be resolved during the catalytic cycle of enzymes such as arsenate reductase. In E.coli, either the thioredoxin or the glutaredoxin pathway can be disabled by mutation without serious
      detriment to the cell. Nevertheless, if both pathways are disrupted, aerobic growth is almost completely eliminated,
      suggesting overlap between these two systems for the reduction of essential substrates such as ribonucleotide
      reductase. However, for some substrates, such as methionine sulfoxide reductase, an apparent specificity towards thioredoxin 1 was deduced from the phenotype of trxA mutants.
• <a href="http://www.pnas.org/cgi/content/full/96/24/13703">Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13703-8</a>.
  • Efficient folding of proteins with multiple disulfide bonds in the E. coli cytoplasm.
  • Under physiological conditions, the E. coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins.
  • However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.
• <a href="http://www3.interscience.wiley.com/cgi-bin/abstract/106567427/ABSTRACT?CRETRY=1&SRETRY=0">Biotechnology and Bioengineering, 85(2):122 - 129, 2003</a>.
  • Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of E. coli.
  • a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system.
  • Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions.
  • The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 g/mL of active urokinase protease in a simple batch reaction.
  • This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.
• <a href="http://www.freepatentsonline.com/20030219870.html">Secretion of proteins with multiple disulfide bonds in bacteria and uses thereof</a> (2003 patent).
  • The invention provides methods for using the Twin Arginine Translocation pathway in bacteria to produce heterologous polypeptides that have multiple disulfide bonds.
  • Methods of screening polypeptide libraries produced by secretion through the TAT pathway are also provided.
  • The invention provides improved methods for production of heterologous polypeptides having at least one disulfide bond.
  • the heterologous polypeptide is secreted from the bacteria and is isolatable from the periplasm of the bacteria or is an integral membrane protein.
  • the invention provides a bacteria genetically transformed with an expression cassette comprising a leader peptide that directs protein export through the Twin Arginine Translocation pathway upstream of a gene encoding a heterologous polypeptide, and wherein the heterologous polypeptide is produced by the bacterial cell and comprises at least one disulfide bond. The bacteria may have an oxidizing cytoplasm. In certain embodiments of the invention, the heterologous polypeptide may contain from about 1 to about 17 disulfide bonds and/or may be produced in biologically-active form. In certain further embodiments of the invention, the leader peptide may be from a gene encoding a protein selected from the group consisting of E. coli TorA, SufI, YacK, YdhX, YdcG, WcaM, YcdB, YaeI, HyaA, HybO, HybA, NapG, NrfC, YagT, YdhX, BisZ, NapA, DmsA, YnfE, YnfF, FdnG, FdoG, YahJ, AmiA, AmiC, YcdB, YedY, FhuD and YaeI. The leader peptide may also be derived from a gene encoding a homologue of any of these sequences.
  • The Tat pathway was discovered less than five years ago as a new mechanism for protein secretion, first in the thylakoid membranes of photosynthetic organisms and subsequently in bacteria. This secretion mechanism has been named the "Twin Arginine Translocation" or TAT pathway because of the signature Arg-Arg motif found in the leader peptides of proteins that are engaged in this mode of export. Estimates based on proteomics and bioinformatics analyses indicate that 5-8% of the secreted proteins in bacteria such as E. coli or B. subtilis are translocated via the TAT pathway.
  • polypeptides exported by the sec pathway and other secretion mechanisms thread the membrane in an unfolded form and reach their native conformation after export is complete, proteins secreted via the TAT pathway first fold in the- cytoplasm and are then translocated across the membrane in a compact, native-like state. In fact, for this reason the TAT pathway is responsible for the export of proteins that require the incorporation of cofactors or the assembly of different subunits in the cytoplasm. Secretion does not occur through the SecYEG translocon but rather through a presumably larger diameter pore formed by the TatABC and E proteins. Large proteins of molecular weight up to at least 120 kDa have been documented to b e exported though the TAT pathway. Moreover, secretion through the TAT pathway is not linked to the hydrolysis of ATP but instead is driven by the asymmetric distribution of protons across the membrane, or .DELTA.pH.
  • The leader peptides required for targeting proteins to the TAT apparatus are longer and less hydrophobic than sec-specific leaders. TAT-specific leader peptides are on average 14 amino acids longer due to an extended amino terminal region and more basic residues in the c-region. However, the hydrophobic region in the TAT-specific leader peptides is significantly shorter due to a higher occurrence of glycine and threonine residues. A hallmark of both plant and prokaryotic TAT-specific leader peptides is the presence of the distinctive and conserved (S/T)-R--R-x-F-L-K sequence motif which is absent from sec-specific leader peptides. This sequence motif is located at the amino terminal region/hydrophobic core boundary within leader peptides of known and predicted TAT substrates. Mutation of either arginine residue within the signal peptide significantly reduces the efficiency of protein translocation.
  • The twin-arginine (RR) motifs of wheat pre-23K and pre-Hcf136 are essential for targeting by the thylakoid TAT pathway. This motif is a central feature of TAT leader peptide in general. Bacterial twin-arginine-signal peptides are similar to thylakoid TAT signals and can direct TAT-dependent targeting into plant thylakoids with high efficiency. However, the vast majority of bacterial signal peptides contain conserved sequence elements in addition to the twin-arginine motif that imply special functions. There is a heavy bias towards phenylalanine at the second position after the twin-arginine motif, and many of the signals contain lysine at the fourth position. None of the known thylakoid twin-arginine signals contains phenylalanine at this position and only one (Arabidopsis P29) contains lysine as the fourth residue after the twin-arginine motif. The precise roles of these highly conserved features are unclear. The phenylalanine residue can be replaced by Leu but not by Ala without undue effects, which indicates that hydrophobicity, rather than the phenylalanine side-chain, might be the important determinant. Similarly, replacement of the Lys residue does not impede export.
  • As shown in Bessette et al., E. coli depends on aerobic growth in the presence of either of the two major thiol reduction systems: the thioredoxin and the glutathione-glutaredoxin pathways. Both the thioredoxins and the glutaredoxins are maintained in a reduced state by the action of thioredoxin reductase (TrxB) and glutathione, respectively. Glutathione is synthesized by the gshA and gshB gene products. The enzyme glutathione oxidoreductase, the product of the gor gene, is required to reduce oxidized glutathione and complete the catalytic cycle of the glutathione-glutaredoxin system. When both of these thiol reduction pathways were eliminated by mutation in a trxB gor or trxB gshA double mutant, the cells grew extremely slowly. However, these cells can be rescued by the addition of the reductant DTT to the growth medium. When a trxB gor or trxB gshA strain was grown in media containing DTT and then transferred to medium lacking DTT, the cytoplasm became even more oxidizing than in the trxB strain. Even when grown in the presence of DTT, both the trxB gshA and trxB gor strains gave rise to fast growing derivatives at a high frequency. Because the trxB, gshA, and gor alleles in these strains are nonreverting null mutations, the faster-growing derivatives must result from extragenic suppressor mutations.
  • these faster-growing derivatives accumulated suppressor mutations in the alkyl hydroperoxidase (ahpC) gene. The resulting ahpC* allele allows efficient growth in normal (non-reducing) media without compromising the formation of disulfide bonds in the cytoplasm. Thus trxB, gor ahpC* mutant strains (such as E. coli DR473 or FA113) exhibit the ability to support disulfide bond formation in the cytoplasm, and also can grow equally well as the corresponding wild-type strain DHB4 in both rich and minimal media. In the studies described herein, the E coli strain DR473-A also contains a mutation that inactivates the periplasmic oxidase dsbA. In addition, DR473-B is a dsbB derivative of DR473. DsbB normally serves as the oxidant of DsbA, and mutants deficient in either enzyme are impaired in the oxidation of proteins in the periplasmic space. Such mutants can form disulfides in the cytoplasm but not in the periplasm.
  • transcriptional fusions between the TAT-specific signal sequence of E. coli trimethylamine N-oxide reductase (TorA) and various genes encoding multidisulfide proteins. These proteins include: (a) E. coli alkaline phosphatase (PhoA) containing two disulfide bonds that are consecutive in the primary sequence (PhoA is a common reporter for sec pathway secretion a s well as disulfide bond formation in vivo); (b) an anti-digoxin antibody fragment (Fab) containing four intra- and one inter-molecular disulfide bond; (c) a truncated version of human tissue plasminogen activator (vtPA) consisting of the kringle 2 and protease domains with a total of nine disulfide linkages; and (d) variants of the E. coli thioredoxin (TrxA). It is shown that the latter protein when expressed in the cytoplasm of the oxidizing strain DR473 and exported via the TAT pathway can serve as a general oxidant within the periplasm. In this case, TrxA that has been pre-oxidized in the cytoplasm and secreted into the periplasm via the TAT pathway can complement dsbB mutants deficient in the normal pathway for the formation of disulfide bonds in the periplasmic space. The export of pre-oxidized thioredoxin variants serves to provide oxidants in the bacterial periplasm and to thus restore defects caused by the lack of normal periplasmic bacterial oxidants such as the enzymes DsbA and DsbB. Defects of dsbA or dsbB mutants that are restored by the oxidized thioredoxin include cell motility, growth in minimal media and infectivity by filamentous phages.
  • fused to the TorA leader peptide.
• <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1472356">Appl Environ Microbiol. 2006 May; 72(5): 3797–3801</a>.
  • Improvement of an Unusual Twin-Arginine Transporter Leader Peptide by a Codon-Based Randomization Approach.
  • Secretion of E. coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value).
  • Recently, a method (called TatP) to predict twin-arginine signal peptides was described. TatP was designed to potentially predict variant Tat signal peptides not containing the consensus twin-arginine motif (Ser/Thr Arg Arg X Phe Leu Lys). Unfortunately, TatP was unsuccessful in identifying the wild-type signal peptide or any of the functional variants described herein as a twin-arginine signal peptide.
• <a href="http://www.biomedcentral.com/1471-2105/6/167">BMC Bioinformatics. 2005 Jul 2;6:167</a>.
  • Prediction of twin-arginine signal peptides.
  • Proteins carrying twin-arginine (Tat) signal peptides are exported into the periplasmic compartment or extracellular environment independently of the classical Sec-dependent translocation pathway.
  • To complement other methods for classical signal peptide prediction we here present a publicly available method, TatP, for prediction of bacterial Tat signal peptides.
  • RESULTS: We have retrieved sequence data for Tat substrates in order to train a computational method for discrimination of Sec and Tat signal peptides. The TatP method is able to positively classify 91% of 35 known Tat signal peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than a complementary rule based prediction method.
  • CONCLUSION: The method developed here is able to discriminate Tat signal peptides from cytoplasmic proteins carrying a similar motif, as well as from Sec signal peptides, with high accuracy. The method allows filtering of input sequences based on Perl syntax regular expressions, whereas hydrophobicity discrimination of Tat- and Sec-signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at <a href="http://www.cbs.dtu.dk/services/TatP/">http://www.cbs.dtu.dk/services/TatP/</a>.
• <a href="http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17597885">Bioinformation. 2006 Jul 25;1(5):184-7</a>.
  • TATPred: a Bayesian method for the identification of twin arginine translocation pathway signal sequences.
  • The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name.
  • There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor.
  • The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.
• <a href="http://www.jbc.org/cgi/content/full/282/11/7903">J Biol Chem. 2007 Mar 16;282(11):7903-11</a>.
  • E. coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.
  • The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.
• <a href="http://www.jbc.org/cgi/content/full/282/11/8309">J Biol Chem. 2007 Mar 16;282(11):8309-16</a>.
  • Export pathway selectivity of E. coli twin arginine translocation signal peptides.
  • The E. coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.
• <a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-4MT5K7Y-F&_user=10&_coverDate=03%2F30%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f280f98f3cf27dfe698a681c0ce145ca">J Mol Biol. 2007 Mar 30;367(3):715-30</a>.
  • An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway.
  • All secreted proteins in E. coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.
• <a href="http://www.springerlink.com/content/b01011416272t583/">Appl Microbiol Biotechnol. 2007 Aug;76(1):35-45</a>.
  • The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production.
  • The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding.
  • The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.
• <a href="http://www.springerlink.com/content/256p780052q1520n/">Appl Microbiol Biotechnol. 2007 Sep;76(3):633-42</a>.
  • Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria.
  • In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein.
• <a href="http://www.proteinscience.org/cgi/content/full/16/5/1001">Protein Sci. 2007 May;16(5):1001-8.</a>
  • A bacterial two-hybrid system based on the twin-arginine transporter pathway of E. coli.
  • We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.
• <a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-4J01YJD-4&_user=10&_coverDate=03%2F17%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=584df8ea5201b35c4b172e87694608f1">J Mol Biol. 2006 Mar 17;357(1):49-61</a>.
  • Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of E. coli: evidence that chaperone activity is the prime effect of thioredoxin.
  • In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of E. coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.
• <a href="http://sciencelinks.jp/j-east/article/200624/000020062406A0940651.php">J Biosci Bioeng. 2006 Oct;102(4):333-9</a>.
  • Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in E. coli trxB and gor double mutant.
  • The dengue virus is currently the most important flavivirus causing human diseases in the tropical and subtropical regions of the world. The envelope protein domain III of dengue virus type 2 (D2EIII), which induces protective and neutralizing antibodies, was expressed as an N-terminal fusion to a hexa-histidine tag in E. coli.
  • The expression of recombinant D2EIII of 103 amino acids in the soluble form can be achieved using suitable host strains, such as Origami, at a low induction temperature of 18 degrees C. The enhanced production of the soluble protein could be attributed to the thioredoxin reductase (trxB) and glutathione reductase (gor) double mutations in the Origami genome.
  • The soluble and refolded D2EIII proteins were recognized by different antibodies including human patient antiserum. The immunization of rats with soluble D2EIII protein elicited the production of antibodies that could recognize the D2EIII protein in the D2EIII precursor protein and in C-terminal truncated dengue envelope protein type 1-4.
  • Thus, this protein production system is suitable for the production of authentic recombinant dengue proteins that may be used in the diagnosis of the dengue virus infection or in vaccine development.
• <a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17502280&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum">J Biosci Bioeng. 2007 Apr;103(4):373-6.</a>
  • Evidence for an additional disulfide reduction pathway in E. coli.
  • An E. coli cell-free protein synthesis cell extract has been created that lacks all known cytoplasmic disulfide reduction pathways but still retains significant reductase activity. Oxidized glutathione was partially stabilized by deleting the gene for glutathione reductase. To avoid previously reported AhpC mutations, thioredoxin reductase was only removed after extract preparation. The trxB gene was extended to encode a hemagglutinin tag so that TrxB could be removed by affinity adsorption. However, significant glutathione reductase activity remained. The unknown glutathione reductase pathway is disabled by iodoacetamide, is inhibited by NADH, and appears to use NADPH as an electron source.
• <a href="http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WPJ-4MJSCGP-3&_user=10&_coverDate=05%2F31%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5b38d73409a7a6fb7922f07158fdeb77">Protein Expression and Purification, 53(1): 124-131, 2007</a>.
  • High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the E. coli cytoplasm for solution NMR studies.
  • a method for the expression in E. coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His₆thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation.
  • When cultured at 37 °C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His₆thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy.
  • This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.
• <a href="http://www3.interscience.wiley.com/cgi-bin/abstract/113510389/ABSTRACT?CRETRY=1&SRETRY=0">Biotechnol Bioeng. 2007 Jul 1;97(4):901-8</a>.
  • Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source.
  • The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50  IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines. 
• <a href="http://www.merckbio.com.tw/molebio.asp?pgurl=molebio.asp&tp1id=04&tpid=0411">Competent Cells</a> from <a href="http://www.merckbio.com.tw">www.merckbio.com.tw</a>
  • Origami
  • Origami B
  • Rosetta-gami-pLysS