Signal Peptide For Protein Secretion Research Articles

Transcriptome Analysis of Fusarium-Tomato Interaction Based on an Updated Genome Annotation of Fusarium oxysporum f. sp. lycopersici Identifies Novel Effector Candidates That Suppress or Induce Cell Death in Nicotiana benthamiana.

Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (secreted in xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast‐growing and unlike yeast, posttranslational modifications like N‐glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non‐activated carbon‐hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α‐promoter. The success of the heterologous production of full‐length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase‐specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

Another Breaker of the Wall: the Biological Function of the Usp45 Protein of Lactococcus lactis.

Lactococcus lactis is a Gram-positive bacterium that is widely used as a cell factory for the expression of heterologous proteins that are relevant in the pharmaceutical and nutraceutical fields. The signal peptide of the major secreted protein of L. lactis, Usp45, has been employed extensively in engineering strategies to secrete proteins of interest. However, the biological function of Usp45 has remained obscure despite more than 25 years of research. Studies on Usp45 homologs in other Gram-positive bacteria suggest that Usp45 may play a role in cell wall turnover processes. Here, we show the effect of inactivation and overexpression of the usp45 gene on L. lactis growth, phenotype, and cell division. Our results are in agreement with those obtained in streptococci and demonstrate that the L. lactis Usp45 protein is essential for proper cell division. We also show that the usp45 promoter is highly activated by galactose. Overall, our results indicate that Usp45 mediates cell separation, probably by acting as a peptidoglycan hydrolase.IMPORTANCE The cell wall, composed mainly of peptidoglycan, is key to maintaining the cell shape and protecting the cell from bursting. Peptidoglycan degradation by peptidoglycan hydrolysis and autolysins occurs during growth and cell division. Since peptidoglycan hydrolases are important for virulence, envelope integrity, and regulation of cell division, it is valuable to investigate their function and regulation. Notably, PcsB-like proteins such as Usp45 have been proposed as new targets for antimicrobial drugs and could also be target for the development of food-grade suicide systems. In addition, although various other expression and secretion systems have been developed for use in Lactococcus lactis, the most-used signal peptide for protein secretion in this bacterium is that of the Usp45 protein. Thus, elucidating the biological function of Usp45 and determining the factors affecting its expression would contribute to optimize several applications.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA ( gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease.

Mining the secretome of the root‐knot nematode Meloidogyne chitwoodi for candidate parasitism genes

Parasite proteins secreted at the interface of nematode and host are believed to play an essential role in parasitism. Here, we present an efficient pipeline of bio-informatic algorithms and laboratory experiments to identify candidate parasitism genes within nematode secretomes, i.e. the repertoire of secreted proteins in an organism. We performed our approach on 12 218 expressed sequence tags (ESTs) originating from three life stages of the plant parasitic nematode Meloidogyne chitwoodi--a molecularly unexplored root-knot nematode species. The ESTs from M. chitwoodi were assembled into 5880 contigs and open reading frames translated from the consensus sequences were searched for features of putative signal peptides for protein secretion and trans-membrane regions, resulting in the identification of 398 secretome members. The products of parasitism genes are secreted by a range of organs, including the oesophageal, amphidial and rectal glands, the intestine, and the hypodermis. To localize the site of expression in M. chitwoodi, we subjected the most abundant secretome members to in situ hybridization microscopy. We found hybridization of one tag in the dorsal oesophageal gland, seven in the two subventral oesophageal glands, two in the intestine and one tag hybridized to the tail tip in the proximity of the phasmids. Four sequences showed similarity to putative parasitism genes from other nematode species, whereas seven represented pioneering sequences. Our approach presents an efficient method to identify candidate parasitism genes, which does not require sophisticated cDNA isolation and selection protocols, and can therefore be used as a powerful starting point for the molecular investigation of parasites.

Comparison of different signal peptides for protein secretion in nonlytic insect cell system

Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.

Molecular cloning of a cDNA clone for tobacco lipid transfer protein and expression of the functional protein in Escherichia coli

A cDNA clone encoding a lipid transfer protein (LTP) was isolated from tobacco by screening a library with a PCR-amplified spinach LTP gene, DNA sequence analysis showed a large open reading frame (344 bp) encoding a polypeptide of 114 amino acids. The first 23 amino acids of the deduced protein have the characteristics of a signal peptide for protein secretion or targeting into dense microbody-like vesicles. The cDNA clone was then inserted into an expression vector, pMAL, and expressed in E. coli as a fusion with the maltose binding protein (MBP). The MBP-LTP fusion protein was purified to homogeneity and subjected to factor Xa cleavage to yield the LTP domain. A lipid transfer assay demonstrated that the resulting LTP was functional. The availability of the expression system in E. coli will facilitate the elucidation of in vivo function(s) of plant LTPs.

Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process.

Subtilisin E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein. The pre-sequence functions as the signal peptide for protein secretion across the membrane. Deletion of the pro-sequence yields mature but inactive subtilisin: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer. We have constructed an active-centre mutant of pro-subtilisin (Asp 32----Asn) which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.

Signal Peptide for Protein Secretion Directing Glycophospholipid Membrane Anchor Attachment

Decay accelerating factor (DAF) is anchored to the plasma membrane by a glycophospholipid (GPI) membrane anchor covalently attached to the COOH-terminus of the protein. A hydrophobic domain located at the COOH-terminus is required for anchor attachment; DAF molecules lacking this domain are secreted. Replacement of the COOH-terminal hydrophobic domain with a signal peptide that normally functions in membrane translocation, or with a random hydrophobic sequence, results in efficient and correct processing, producing GPI-anchored DAF on the cell surface. The structural requirements for GPI anchor attachment and for membrane translocation are therefore similar, presumably depending on overall hydrophobicity rather than specific sequences.

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli.

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.