Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98–114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98–114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

Antimicrobial peptides from C-terminal amphipathic region of E. coli FtsA

Antimicrobial peptides constitute an indispensable component of innate immune system in organisms ranging from bacteria to man. Despite this, peptides lag far behind the conventional antibiotics in treating infections. The menace of multidrug-resistant bacteria, however, has revived the antimicrobial peptide research. We reasoned that the membrane-binding regions of bacterial proteins could be purposed to combat them. Here, we identify potent antimicrobial peptides from the C-terminal amphipathic helix of E. coli FtsA protein. The 11 and 13-residue peptides exhibited activity against E. coli, gentamicin-resistant MRSA, and C. albicans. The activity is little affected by the presence of salt and divalent cations. The peptides preferentially bind to the negatively-charged membranes as indicated by tryptophan fluorescence studies. The peptides permeabilize the E. coli outer and inner membranes at very promising concentrations suggesting membrane-disruption as one of the mechanisms of killing.     

Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.     

Structure–activity relationship of potent antimicrobial peptide analogs of Ixosin-B amide

There is a great urgency in developing a new generation of antibiotics and antimicrobial agents since the bacterial resistance to antibiotics have increased dramatically. A series of overlapped peptide fragments of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, was designed, synthesized and examined for their antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A potent 11-mer peptide TSG-8-1, WWSYVRRWRSR-amide, was developed, which exhibited antimicrobial activity against E. coli and S. aureus while very little hemolytic activity in human erythrocytes was observed at high dose level. This peptide could be further modified for the development of a potent antimicrobial agent in the future.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Phages with high affinity to the S protein obtained fromrice stripe virus (RSV) were enriched fromphage-displayed random 12-mer peptide library after threerounds of biopanning. 9 different peptides from theenriched library were selected by ELISA. Circulardichroism (CD) spectra of the GST-S fusion protein withbinding phages and non-binding phages showed thatstructure of the S protein was changed after it bound toeach of these 9 selected 12-mer peptides, which suggestedthat these peptides might disrupt the function of Sprotein. Thus, those peptides might be used to developplant resistance and disrupt virus transmission. 3 of the12-mer peptide genes were fused with the GST gene in pGEX3X. The fusion proteins were also obtained using E.coli expression system and purified.     

Functional whole-colony screening method to identify antimicrobial peptides

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides.Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100 000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.     

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract

Background

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.

Methodology/Principal Findings

Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD₆₂₀ 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10).

Conclusion/Significance

While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.     

Etiology of Childhood Infectious Diarrhea in a Developed Region of China: Compared to Childhood Diarrhea in a Developing Region and Adult Diarrhea in a Developed Region

In China, great differences in economy, social characteristics and hygiene exist between developing and developed regions. A comparative study of infectious diarrhea between two regions was needed. Three groups of diarrheal patients were collected: children ≤5 year-olds from Beijing (developed region) and Henan Province (developing region), and adults over 18 year-olds from Beijing. A questionnaire was used to survey and feces samples were examined for 16 enteropathogens. We enrolled 1422 children and 1047 adults from developed region and 755 children from developing region. Virus positive rates were 32.98% for children and 23.67% for adults in developed region. The most prevalent pathogen for children was rotavirus whereas for adults was norovirus. Bacterial isolation rates were 13.92% for children from developed region, while 29.14% for children from the developing regions. For the greatest difference, Shigella accounted for 50.79% and was the dominant pathogen in the developing region, whereas in the developed region it was only 1.45%. There was no significant relationship between the local levels of development with diarrheogenic Escherichia coli (DEC) categories. But it was seen the notable differences between the population with different age: enteropathogenic E.coli (EPEC) and enteroaggregative E.coli (EAggEC) were the primary classes of DEC in children from both regions, whereas it was enterotoxigenic E.coli (ETEC) in adults. The symptoms of Shigella and Salmonella infection, such as bloody stools, white blood cells (WBC) and red blood cells (RBC) positivity and fever were similar in children, which may lead to the misidentification. Yersinia enterocolitica and shiga toxin-producing E.coli (STEC) infections were firstly reported in Beijing. There was a large difference in etiology of bacterial diarrhea between children in developing and developed regions of China.     

Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.     

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.     

DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2

The araB and araC genes of Salmonella typhimurium have been cloned onto the plasmid pBR322. Restriction analysis and subcloning of restriction fragments localized these genes to a 4.4 kb DNA fragment. Complementation analysis revealed that the cloned araB and araC genes from S. typhimurium complemented araB and araC mutant strains of Escherichia coli. Conversely, cloned araB and araC genes from E. coli complemented araB and araC mutant strains of S. typhimurium. The DNA sequences was determined for the S. typhimurium araB and araC controlling region and for the initially translated portions of these genes. The nucleotide sequence of the araB promoter was 87% homologous with the same region in E. coli and contained no deletions or insertions relative to the E. coli sequence. The presumed AUG codon corresponding to the amino terminus of the S. typhimurium araC protein was in the same location as in E. coli. There was, however, considerable divergence from the E. coli sequence preceding the translation start site. The nucleotide sequence of the initial 237 bp in the open reading frame of the S. typhimurium araC gene was 78% homologous with the same sequence in E. coli. By comparison, the amino acid sequence for this region was 91% conserved.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Synthesis of polyoma proteins in vitro

Polyoma virus DNA can be transcribed and translated in an extract of Escherichia coli. Proteolytic digests of the product contain peptides that correspond to peptides from the virion. One of the in vitro synthesized polypeptides corresponds in size to the major virion protein but most of the product is smaller in size than this protein. Although most of the tryptic peptides corresponding to the major virion protein can be detected in the in vitro synthesized polypeptides, all of them also gave rise to other, non-matching, peptides. Much of the in vitro product therefore appears to correspond to incomplete molecules of the major virion protein.     

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.     

A hybrid bacterial replication origin

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.     

Peptide Ligands That Bind Selectively to Spores of Bacillus subtilis and Closely Related Species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.