Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the -factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg^–1 protein which was more than a recombinant P. pastoris GS115 (552 U mg^–1 protein) or KM71H (539 U mg^–1 protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg^–1 protein by P. pastoris GS115, 1176 U mg^–1 protein by P. pastoris KM71H and 1522 U mg^–1 protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 °C) than the wild-type PLC from B. cereus. Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co²⁺ and Mn²⁺ etc., also influenced the activity of the recombinant PLCs.     

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289–296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria

Summary The two-step protein secretion pathway in Pseudomonas aeruginosa is dependent on the xcp genes. We investigated whether a similar secretion mechanism is present in non-pathogenic Pseudomonas spp. and in other gram-negative bacteria. The plant growth stimulating Pseudomonas strains P. putida WCS358, P. fuorescens WCS374 and Pseudomonas 1310 appeared to secrete proteins into the extracellular medium. Southern hybridization experiments showed the presence of xcp genes in these strains and also in other gram-negative bacteria, including Xanthomonas campestris. Complementation experiments showed that the xcp gene cluster of P. aeruginosa restored protein secretion in an X. campestris secretion mutant. The secretion gene cluster of X. campestris however, restored secretion capacity in P. aeruginosa mutants only to a low degree. Two heterologous proteins were not secreted by P. fuorescens and P. aeruginosa. The results suggest the presence of a similar two-step protein secretion mechanism in different gram-negative bacteria, which however, is not always functional for heterologous proteins.     

A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78

Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant–bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

幽门螺杆菌分子伴侣GroEL相互作用蛋白分析

【目的】获得幽门螺杆菌(Helicobacter pylori,HP) GroEL结合蛋白质组构成谱,为进一步探究GroEL及其与相互作用蛋白在HP致病机制中的作用提供新思路。【方法】在构建HP GroEL原核表达重组大肠杆菌(Escherichia coli) BL21(DE3)⁽pET-28a(+)-groEL)基础上,纯化带有His标签的GroEL蛋白,与HP全菌蛋白提取液共孵育后,利用Protein G磁珠和抗His标签抗体免疫沉淀法对复合物进行捕获,然后对复合物中GroEL及其结合的蛋白质进行质谱法鉴定,根据主要功能对其进行分类,并完成蛋白质相互关系网络分析。【结果】对GroEL蛋白捕获成分进行分析,共鉴定出59种可能与GroEL结合的蛋白质,其中包括19种代谢酶类(KatA、GltA和AhpC等参与氧化还原相关酶类7种,PepA、RocF和HtrA等肽酶5种,以及2种参与脂肪代谢酶、2种参与ATP合成酶、2种尿素酶和HP17＿08079蛋白等)、15种外膜蛋白(黏附素BabA、SabA、HapA及其他膜蛋白等)、8种转录翻译相关蛋白(Tuf、RpoBC...     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals

Human interferon-alpha 2b (IFN-α2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-α2b, Saccharomyces cerevisiae MF-α factor prepro sequence and a mutated α prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-α2b into the culture medium of P. pastoris. The native secretion signal of IFN-α2b did not secrete protein into the culture medium of P. pastoris. The α prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-α2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-α2b secreted by human lymphocytes. The full α prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-α2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full α prepro sequence was used for the secretion of IFN-α2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.     

Tissue-specific position effects on alcohol dehydrogenase expression in Drosophila melanogaster

Summary Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three proteases (PrtA, PrtB and PrtC) into the extracellular medium. The gene encoding the 50 kDa protease, prtA, was subcloned from a recombinant cosmid carrying a fragment of the E. chrysanthemi B374 chromosome. prtA was shown to be located immediately 3 to the structural genes for the other two extracellular proteases. The amino acid sequence of PrtA, as predicted from the prtA nucleotide sequence, showed a high level of homology with a family of metalloproteases that are all secreted via a signal peptide-independent pathway, including PrtB and PrtC of E. chrysanthemi B374, PrtC of E. chrysanthemi EC16, PrtSM of Serratia marcescens and AprA of Pseudomonas aeruginosa. PrtA secretion requires the E. chrysanthemi protease secretion factors PrtD, PrtE and PrtF. The secretion signal of PrtA is near to the carboxy-terminal end of the protein, as was previously shown to be the case for PrtB and PrtSM and for Escherichia coli -hemolysin. The C-termini of these four proteins do not show extensive primary sequence homology, but PrtA, PrtB and PrtSM each have a potential amphipathic -helix located close to the C-terminus.     

Isolation and expression analysis of low temperature-induced genes in white poplar (Populus alba)

Poplar is an important crop and a model system to understand molecular processes of growth, development and responses to environmental stimuli in trees. In this study, we analyzed gene expression in white poplar (Populus alba) plants subjected to chilling. Two forward suppression-subtractive-hybridization libraries were constructed from P. alba plants exposed to low non-freezing temperature for 6 or 48 h. Hundred and sixty-two cDNAs, 54 from the 6-h library and 108 from the 48-h library, were obtained. Isolated genes belonged to six categories of genes, specifically those that: (i) encode stress and defense proteins; (ii) are involved in signal transduction; (iii) are related to regulation of gene expression; (iv) encode proteins involved in cell cycle and DNA processing; (v) encode proteins involved in metabolism and energetic processes; and (vi) are involved in protein fate.Different expression patterns at 3, 6, 12, 24, 48 h at 4 °C and after a recovery of 24 h at 20 °C were observed for isolated genes, as expected according to the class in which the gene putatively belongs. Forty-four of 162 genes contained DRE/LTRE cis-elements in the 5′ proximal promoter of their orthologs in Populus trichocarpa, suggesting that they putatively belong to the CBF regulon. The results contribute new data to the list of possible candidate genes involved in cold response in poplar.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Production of <Emphasis Type="Italic">Chryseobacterium proteolyticum</Emphasis> protein-glutaminase using the twin-arginine translocation pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.     

基于MS2噬菌体lys基因的质粒型条件自溶菌的构建与应用

【背景】传统外源蛋白的原核表达通常需要以超声破碎或者酶解的方式破碎菌体,过程比较烦琐。【目的】构建基于MS2噬菌体lys基因的质粒型条件自溶菌,以简化外源蛋白的获取流程。【方法】从MS2噬菌体中克隆lys基因,构建重组表达质粒,并在大肠杆菌BL21(DE3)中异源表达,以此构建质粒型条件自溶菌,通过生长曲线和菌落形成单位反映自溶菌裂解效率,利用SDS-PAGE检测外源蛋白释放情况。【结果】构建了pBAD-lys BL21(DE3)、pBAD-Opti-lys BL21(DE3)及pCDF-BAD-Opti-lys BL21(DE3)这3种质粒型条件自溶菌。以上自溶菌在阿拉伯糖诱导后其宿主裂解效率均为99.99%以上,CFU结果显示含pCDF-BAD-Opti-lys质粒的宿主裂解效果更优,在此自溶菌BL21(DE3)中表达含His标签的重组绿色荧光蛋白(enhanced green fluorescent protein,eGFP),经阿拉伯糖诱导后菌体中约63.00%以上的eGFP释放至胞外,利用Ni-NTA可以直接从培养基中纯化得到约30 kDa的单一目的蛋白。【结论】基于MS2噬菌体lys基因成功构建了阿拉伯糖诱导的质粒型条件自溶菌,此自溶菌能够以自我裂解的方式释放大部分胞内外源蛋白,简化传统外源蛋白获取流程。