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1.
Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein 总被引:20,自引:0,他引:20
G P Liu T B Topping W H Cover L L Randall 《The Journal of biological chemistry》1988,263(29):14790-14793
We have proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that during export of protein from Escherichia coli, there is a kinetic partitioning between the pathway that leads to productive translocation and the pathway that leads to folding of precursors into a stable conformation that is incompatible with export. This model predicts that a decrease in rate along the productive pathway resulting from a defect in the leader sequence could be partially overcome by slowing the folding of the precursor and thereby increasing the time during which that polypeptide would be competent to enter the export pathway. Here it is shown that a change in the mature portion of maltose-binding protein that is known to suppress a mutation in the leader sequence (Cover, W. H., Ryan, J. P., Bassford, P. J., Jr., Walsh, K. A., Bollinger, J., and Randall, L. L. (1987) J. Bacteriol. 169, 1794-1800) also decreases the rate of folding of the precursor. 相似文献
2.
Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli 总被引:32,自引:0,他引:32
We have examined the effects of thermosensitive mutations in secA and secY (prlA) genes on the export of proteins to the three layers of the Escherichia coli cell surface. After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoprotein and OmpA, is delayed, then essentially blocked, in either a secA or secY strain. These mutations also have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane. However, the membrane assembly of M13 coat protein (an inner membrane protein made with an amino-terminal leader sequence) is not affected. Thus, the requirement for sec function for export does not correlate with the presence or absence of leader peptide or with a particular subcellular compartment, but rather is specific to each particular protein. 相似文献
3.
Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY 总被引:15,自引:0,他引:15
The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides. 相似文献
4.
Angela A. Lilly Jennine M. Crane Linda L. Randall 《Protein science : a publication of the Protein Society》2009,18(9):1860-1868
SecB, a remarkable chaperone involved in protein export, binds diverse ligands rapidly with high affinity and low specificity. Site‐directed spin labeling and electron paramagnetic resonance spectroscopy were used to investigate the surface of interaction on the export chaperone SecB. We examined SecB in complex with the unfolded precursor form of outer membrane protein OmpA as well as with a truncated version of OmpA that includes the transmembrane domain and lacks both the signal peptide and the periplasmic domain. In addition, we studied the binding of SecB to the unfolded mature form of galactose‐binding protein, a soluble periplasmic protein. We have previously used the same strategy to map the binding surface for the precursor of galactose‐binding protein. We show that for all ligands tested the patterns of contact are the same. 相似文献
5.
Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone 总被引:19,自引:0,他引:19
The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase. 相似文献
6.
A gene encoding a hevein-like protein from elderberry fruits is homologous to PR-4 and class V chitinase genes 总被引:1,自引:0,他引:1
Van Damme EJ Charels D Roy S Tierens K Barre A Martins JC Rougé P Van Leuven F Does M Peumans WJ 《Plant physiology》1999,119(4):1547-1556
We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene. 相似文献
7.
Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase 总被引:10,自引:0,他引:10
The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system. 相似文献
8.
The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane 总被引:45,自引:0,他引:45
The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other. 相似文献
9.
Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane 总被引:39,自引:0,他引:39
Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins. We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter. This strain requires arabinose for growth. When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein). These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K. However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane. Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane. 相似文献
10.
The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a precursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationally by the highly conserved translocase Cox18 and associated proteins. We have found that Cox20 is required for efficient export of the Cox2 C-tail. Furthermore, Cox20 interacts by co-immune precipitation with Cox18, and this interaction requires the presence of Cox2. We therefore propose that Cox20 binding to Cox2 on the trans side of the inner membrane accelerates dissociation of newly exported Cox2 from the Cox18 translocase, promoting efficient cycling of the translocase. The requirement for Cox20 in cytochrome c oxidase assembly and respiratory growth is partially bypassed by yme1, mgr1 or mgr3 mutations, each of which reduce i-AAA protease activity in the intermembrane space. Thus, Cox20 also appears to stabilize unassembled Cox2 against degradation by the i-AAA protease. Pre-Cox2 leader peptide processing by Imp1 occurs in the absence of Cox20 and i-AAA protease activity, but is greatly reduced in efficiency. Under these conditions some mature Cox2 is assembled into cytochrome c oxidase allowing weak respiratory growth. Thus, the Cox20 chaperone has important roles in leader peptide processing, C-tail export, and stabilization of Cox2. 相似文献
11.
Stuart C H Allen Claire M L Barrett Nicola Ray Colin Robinson 《The Journal of biological chemistry》2002,277(12):10362-10366
The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export. 相似文献
12.
The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB 总被引:11,自引:0,他引:11
In the accompanying paper (Altman, E., Bankaitis, V.A., and Emr, S.D. (1990) J. Biol. Chem. 265, 18148-18153) a putative SecB binding site was identified in the mature LamB protein. The export of wild-type LamB was unperturbed when this region was removed, however, suggesting the presence of a second site of interaction between SecB and LamB. In this paper we show that the interference caused by export-defective LamB proteins is influenced by the amount of signal sequence that is present. If a large portion of the signal sequence is deleted then the interference levels are significantly reduced. This result suggests that a region of the signal sequence contributes to the interaction of SecB with the LamB protein. Using anti-SecB affinity chromatography, we demonstrated directly that the association of SecB protein with precursor LamB is dependent on the presence of both the LamB signal sequence and the interfering region which maps to amino acids 320-380 of mature LamB. Although the interfering region is not necessary for the export of wild-type LamB under normal conditions, when the signal sequence is mutationally altered the interfering region is required to promote the efficient export of LamB protein. Also, deletion of the interfering region eliminates the ability of wild-type LamB precursor to be maintained in an export competent conformation in vivo. Collectively, our results indicate that efficient export of the LamB protein is achieved by an interaction with SecB that involves both the LamB signal sequence and the interfering region in mature LamB. 相似文献
13.
Pseudomonas solanacearum is an important phytopathogen that produces a variety of extracellular enzymes. Previous reports suggested that one of these, a 43-kDa beta-1,4-endoglucanase (EGL), is initially synthesized with a 45-residue leader sequence that is removed during export. Experiments with globomycin presented here also suggest that the primary precursor of EGL (ppEGL) has a 45-residue leader sequence but that only the first 19 residues of the leader sequence are removed by signal peptidase II during initial export across the inner membrane. Further analysis suggested that the resultant 46-kDa intermediate precursor (pEGL) is a transient fatty acylated lipoprotein and is located on the periplasmic side of the inner membrane of P. solanacearum. Although Escherichia coli could synthesize ppEGL, modify it with palmitate, and remove the first 19 residues of the leader sequence during export across the inner membrane, only P. solanacearum could export pEGL across the outer membrane and remove the remaining 26 residues of the leader sequence producing the mature, extracellular EGL. The second step of the export process requires export machinery not present in E. coli. To our knowledge this represents the first example of a leader sequence with two distinct parts, one removed during export across the inner membrane and the other removed during export across the outer membrane. 相似文献
14.
Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors 总被引:9,自引:0,他引:9
A L Brown L Chiariotti C C Orlowski T Mehlman W H Burgess E J Ackerman C B Bruni M M Rechler 《The Journal of biological chemistry》1989,264(9):5148-5154
The insulin-like growth factors (IGFs), IGF-I and IGF-II, occur in plasma and tissue fluids complexed to specific binding proteins. Although the role of the binding proteins is not completely defined, they are capable of modulating the biological activity of the IGFs. In order to better understand the function of these proteins, we have isolated a clone from the BRL-3A rat liver cell line that encodes a protein corresponding to the IGF binding protein in fetal rat serum. The cDNA clone encodes a precursor protein of 304 amino acids (32,886 daltons), comprised of a 34-residue hydrophobic prepeptide and a 270-residue mature protein (29,564 daltons). The deduced amino acid sequence agrees with the sequence of 173 amino acid residues determined by Edman degradation. The mature protein contains 18 cysteines and no N-glycosylation sites. It contains an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus. A similar sequence is present on many extracellular matrix proteins and contributes to their recognition by cellular adhesion receptors. The cloned cDNA has been transcribed in vitro and the resulting RNA expressed in Xenopus oocytes. Injected oocytes secrete a 33-kDa protein that is immunoprecipitated by polyclonal antibodies to the BRL-3A binding protein and binds IGF-I and IGF-II with the same affinity and specificity as does purified BRL-3A binding protein. The binding protein cDNA probe hybridizes to an approximately 2-kilobase mRNA in BRL-3A cells and in multiple fetal rat tissues including liver, kidney, intestine, and lung. Levels of this mRNA are greatly reduced in the corresponding adult tissues. The rat IGF binding protein is closely related to the partial amino acid sequences reported for a bovine IGF binding protein and more distantly related to a human IGF binding protein that recently has been cloned. No significant homologies were identified to other proteins. Thus, the rat IGF binding protein that we have cloned appears to be a distinct member of a family of related IGF binding proteins. We postulate that the structurally distinct IGF binding proteins may have different biological functions. 相似文献
15.
Efficient in vivo translocation of the precursor of Escherichia coli outer membrane protein PhoE across the inner membrane is shown to depend on SecB protein. A set of mutants, carrying internal deletions in the phoE gene, was used to locate a possible SecB-binding site and/or a site that makes the protein dependent on SecB for export. Except for two small mutant PhoE proteins, the in vivo and in vitro translocation of all mutant proteins was more efficient in the presence of SecB. The interaction of SecB protein with wild-type and mutant PhoE proteins, synthesized in vitro, was further studied in co-immunoprecipitation experiments with anti-SecB protein serum. The efficiencies of co-immunoprecipitation of precursor and mature PhoE were very similar, indicating the absence of a SecB-binding site in the signal sequence. Moreover, all mutant proteins with deletions in the mature moiety of the PhoE protein were co-immunoprecipitated in these assays, albeit mostly with reduced efficiency. Taken together, these results indicate the existence of multiple SecB-binding sites in the mature portion of the PhoE protein. 相似文献
16.
D. R. Blanco M. Giladi C. I. Champion D. A. Haake G. K. Chikami J. N. Miller M. A. Lovett 《Molecular microbiology》1991,5(10):2405-2415
Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites. 相似文献
17.
Mutations that affect the folding of ribose-binding protein selected as suppressors of a defect in export in Escherichia coli 总被引:8,自引:0,他引:8
C M Teschke J Kim T Song S Park C Park L L Randall 《The Journal of biological chemistry》1991,266(18):11789-11796
It has been proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export. As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export. (Liu, G., Topping, T. B., Cover, W. H., and Randall, L. L. (1988) J. Biol. Chem. 263, 14790-14793). Here we describe mutations of the gene encoding ribose-binding protein that were selected as suppressors of a defect in export of that protein and that alter the folding pathway. We propose that selection of such suppressors may provide a general method to obtain mutations that affect the folding properties of any protein that can be expressed and exported in E. coli. 相似文献
18.
Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. 总被引:20,自引:1,他引:19
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The complete amino acid sequence of CRP55, the major 55 kd calcium binding protein of the ER lumen, was deduced from the murine cDNA nucleotide sequence. This was completed using a novel application of PCR amplification. The mature 399 residue protein encoded is preceded by a 17 amino acid leader sequence and ends in the ER signal sequence, KDEL. The protein contains no calcium binding motifs of the EF hand type or of the form seen in calelectrin-related proteins. The major region of potential low affinity calcium binding sites is a polyacidic stretch towards the C terminus. The primary structure of the protein is markedly zonal. The N-terminal region, of approximately neutral net charge and hydrophobicity, is followed by a central proline-rich zone with repeat sequences separated from the polyacidic C-terminal stretch by a short hydrophobic sequence. The general shape suggested is a globular domain attached to an extended tail. Immunofluorescence studies show that the protein is present in skeletal muscle and indicate that it is a sarcoplasmic reticulum protein. We propose that the protein be named calreticulin to reflect its calcium binding activity and location in the ER and SR. 相似文献
19.
Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.
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It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane. 相似文献
20.
Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria. 相似文献