Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part I: overview

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In this review, we discuss the exploitation of such fermentation towards the development of functional foods conferring specific health benefits to the consumer beyond basic nutrition. In particular, in Part I, we focus on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of LAB as cell factories for the de novo generation of bioactivities.     

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.     

The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.     

Protein-fold evolution in the test tube

Currently, the combination of library selection and directed evolution is the most powerful approach for finding proteins with novel folds or functions. In the past, most studies concentrated either on protein scaffolds with a given fold or on short peptides. With the recent development of potent in vitro selection and evolution techniques, the screening of much larger sequence space is possible, allowing for the de novo generation of proteins.     

弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究

家蝇抗菌肽多是碱性蛋白,目前尚无弱酸性家蝇抗菌肽的报道。通过稀醋酸低温浸提,海藻酸吸附,稀盐酸低温洗脱、盐析、Sephadex G25凝胶过滤和CMC23弱阳离子交换柱层析等方法,利用灵敏的杀菌活性检测手段,从家蝇蛆(Musca domesticalarvae)中分离纯化出一组弱酸性抗菌肽,对苏云金芽孢杆菌(Bacillus thuringiensis)等革兰氏阳性菌和几种革兰氏阴性菌有强烈的杀灭作用,有极强的耐热、耐冻融的特性。通过电洗脱方法进一步纯化出抗菌肽MD7095,质谱测定其分子量7095Da,IEF电泳测得其等电点5.59,经肽质量指纹谱(PMF)鉴定为一新肽。扫描电镜超微结构观察表明,弱酸性家蝇蛆抗菌肽对苏云金芽孢杆菌的杀菌机制主要是使细胞膜穿孔,内容物外泄,最终使细菌完全解体死亡。     

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix–loop–helix peptides

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix–loop–helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.     

Improving performance of mammalian cells in fed-batch processes through "bioreactor evolution"

The amount of recombinant product obtained from mammalian cells grown in a bioreactor is in part limited by achievable cell densities and the ability of cells to remain viable over extended periods of time. In an attempt to generate cell lines capable of better bioreactor performance, we subjected the DG44 Chinese Hamster Ovary (CHO) host cell line and a recombinant production cell line to an iterative process whereby cells capable of surviving the harsh conditions in the bioreactor were selected. This selective process was termed "bioreactor evolution". Following the selective process, the "evolved" host cells attained a 2-fold increase in peak cell density and a 72% increase in integral cell area. Transient transfection experiments demonstrate that the evolved cells have the same transfection efficiency and the same secretory potential as the initial cells. The "evolved" host was also found to contain a large subpopulation of cells that did not require insulin for growth. From this, a new population of growth-factor-independent cells was obtained. These improvements in host properties should prove beneficial in the expression of recombinant proteins in fed-batch processes. The selective process was also applied to a recombinant production cell line. The evolved cells from this selection exhibited a 38% increase in peak cell density, a 30% increase in integral cell area, and a 36% increase in product titer. These increases were obtained without any appreciable impact on product quality, demonstrating the usefulness of this simple approach to improve the performance of recombinant cell lines.     

Lividins: novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida. Identification by "shotgun" cDNA cloning and sequence analysis

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a "shotgun" skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1-4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.     

A combined mass spectrometric and cDNA sequencing approach to the isolation and characterization of novel antimicrobial peptides from the skin secretions of Phyllomedusa hypochondrialis azurea

Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent anti-microbial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. 3'-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 microM when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus.     

Sequence similarity-driven proteomics in organisms with unknown genomes by LC-MS/MS and automated de novo sequencing

LC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, combined with data processing, stringent, and sequence-similarity database searching tools, was employed in a layered manner to identify proteins in organisms with unsequenced genomes. Highly specific stringent searches (MASCOT) were applied as a first layer screen to identify either known (i.e. present in a database) proteins, or unknown proteins sharing identical peptides with related database sequences. Once the confidently matched spectra were removed, the remainder was filtered against a nonannotated library of background spectra that cleaned up the dataset from spectra of common protein and chemical contaminants. The rectified spectral dataset was further subjected to rapid batch de novo interpretation by PepNovo software, followed by the MS BLAST sequence-similarity search that used multiple redundant and partially accurate candidate peptide sequences. Importantly, a single dataset was acquired at the uncompromised sensitivity with no need of manual selection of MS/MS spectra for subsequent de novo interpretation. This approach enabled a completely automated identification of novel proteins that were, otherwise, missed by conventional database searches.     

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.     

一种用于穿透多肽筛选的随机文库的构建及筛选

以增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)为示踪物,在pET-14b载体上构建编码12个氨基酸的随机多肽表达文库.建立一种简便、经济、有效的文库筛选方法,从所构建的文库中筛选出细胞穿透多肽（cell-penetrating peptide, CPP）. 采用点突变技术,首先在pET-14b载体多克隆位点NdeⅠ和XhoⅠ之间加入4个限制性内切酶位点,随后在BamH Ⅰ位点后加入三联终止密码子,接着再利用亚克隆的方法在Kpn Ⅰ 和XhoⅠ之间插入EGFP,形成一个新的用于原核表达示踪蛋白的载体pET-14bMCStop/EGFP.最后再利用点突变技术在上述构建的示踪载体的多克隆位点XhoⅠ和BamH Ⅰ之间插入36个随机碱基序列.以His-Tat-EGFP作为工具建立有效的筛选方法,利用这种方法对文库进行筛选. 酶切和测序表明,示踪载体的构建是正确的,且在大肠杆菌中可有效地表达出His标记的EGFP.在示踪载体的基础上构建的随机多肽文库至少包含了10⁵个独立克隆,其中90%以上的克隆插入的随机片段都是36个碱基.建立的筛选方法是可行的,并用此方法进行了初步的筛选.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Rice dehydrin K-segments have <Emphasis Type="Italic">in vitro</Emphasis> antibacterial activity

Dehydrins are groups of plant proteins that have been shown to response to various environmental stimuli such as dehydration, elevated salinity, and low temperature. However, their roles in plant defense against microbes have not been demonstrated. In an attempt to discover plant antimicrobial proteins, we have screened a rice cDNA library and isolated several cDNAs coding for dehydrins. Protein extracts from Escherichia coli expressing these cDNAs were tested for their activity against Gram-positive bacteria (Bacillus pumilus, B. subtilis, Staphylococcus aureus, and Sarcina lutea) and Gramnegative bacteria (Escherichia coli and Xanthomonas oryzae pv. oryzae). The results indicate that the crude protein extracts exhibited antibacterial activities against the Gram-positive bacteria. However, dehydrins purified by immunoaffinity chromatography were not active against the bacteria. To pinpoint the dehydrin peptides that were responsible for the bactericidal activity, we expressed DNA sequences coding for truncated dehydrins containing either K- or S-segment and found that K-segment peptides, and not S-segment, were responsible for the antibacterial activities against Gram-positive bacteria. Antibacterial assay with synthetic K-segments indicated that the peptides inhibited growth of B. pumilus with minimum inhibition concentration and minimum bactericidal concentration of 130 and 400 μg/ml, respectively.     

DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: "maintenance" and "de novo" activities are carried out by the same enzyme molecule

DNA-5-methyltransferase has been purified (about 1400-fold) from rapidly proliferating mouse P815 mastocytoma cells by chromatographies on DEAE cellulose, hydroxyapatite and a heparine-agarose affinity step. The isolated enzyme has an isoelectric point of 7.3 and in neutral 10-30% glycerol gradient it bands in an area corresponding to molecular weight of 135,000 dalton. During the enzymatic reaction, the enzyme first interacts with DNA and then accomplishes a series of methyl group transfers without being detached. The formation of the initial DNA-enzyme complexes is probably random and independent of the cofactor, S-adenosyl-L-methionine, as well as the sequences recognized as methylation sites. The "maintenance" and "de novo" types of activity have been monitored using hemimethylated and completely unmethylated DNA as methyl group accepting polymers. Both these activities copurify in three different chromatographic procedures. This, together with the fact that the enzyme purified near to homogeneity possesses both types of activities suggests that "de novo" and "maintenance" DNA methyltransferase activities are exercised by the same enzyme molecule.     

De novo heme proteins from designed combinatorial libraries.

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.     

Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells

The concentration of uridine in the media of cultured L1210 cells was maintained within the concentration range found in plasma (1 to 10 microM) to determine if such concentrations are sufficient to satisfy the pyrimidine requirements of a population of dividing cells and to determine if cells utilize de novo and/or salvage pathways when exposed to plasma concentrations of uridine. When cells were incubated in the presence of N-(phosphonacetyl)-L-aspartate to block de novo biosynthesis, plasma concentrations of uridine maintained normal cell growth. De novo pyrimidine biosynthesis, as determined by [14C]sodium bicarbonate incorporation into uracil nucleotides, was affected by the low concentrations of uridine found in the plasma. Below 1 microM uridine, de novo biosynthesis was not affected; between 3 and 5 microM uridine, de novo biosynthesis was inhibited by approximately 50%; and above 12 microM uridine, de novo biosynthesis was inhibited by greater than 95%. Inhibition of de novo biosynthesis correlated with an increase in the uracil nucleotide pool. The de novo pathway was much more sensitive to the uracil nucleotide pool size than was the salvage pathway, such that when de novo biosynthesis was inhibited by greater than 95% the uracil nucleotide pool continued to expand and the cells continued to take up [14C]uridine. Thus, the pyrimidine requirements of cultured L1210 cells can be met by concentrations of uridine found in the plasma and, when exposed to such physiologic concentrations, L1210 cells decrease their dependency on de novo biosynthesis and utilize their salvage pathway. Circulating uridine, therefore, may be of physiologic importance and could be an important determinant in anti-pyrimidine chemotherapy.