Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase

The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptide surfactin, a highly potent biosurfactant synthesized by a large multimodular peptide synthetase. We report the genetic engineering of the surfactin biosynthesis resulting in the production of a novel lipohexapeptide with altered antimicrobial activities. A combination of in vitro and in vivo recombination approaches was used to construct a modified peptide synthetase by eliminating a large internal region of the enzyme containing a complete amino acid incorporating module. The remaining modules adjacent to the deletion were recombined at different highly conserved sequence motifs characteristic of amino acid incorporating modules of peptide synthetases. The primary goal of this work was to identify permissive fusion sites suitable for the engineering of peptide synthetase genes by genetic recombination. Analysis of the rearranged enzymes after purification from B. subtilis and from the heterologous host Escherichia coli revealed that the selection of the recombination site is of crucial importance for a successful engineering. Only the recombination at a specific HHII x DGVS sequence motif resulted in an active peptide synthetase. The expected lipohexapeptide was produced in vivo and first evidence of a reduced toxicity against erythrocytes and an enhanced lysis of Bacillus licheniformis cells was shown.     

Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli

Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-terminal homoserine lactone (DCD-1Hsl). Dermcidin coding sequence was cloned downstream of a 125 amino acid ketosteroid isomerase gene and upstream of a His6Tag sequence in pET-31b(+) vector and transformed into Escherichia coli. The fusion protein was expressed in the form of inclusion bodies, purified on His Bind Resin, and cleaved by CNBr to release recombinant DCD-1Hsl. Purification of rDCD-1Hsl was achieved by solid-phase extraction that yielded milligram amounts of peptide with more than 95% purity. Recombinant peptide showed antimicrobial activities against E. coli ML-35p, Salmonella typhimurium 5156, Listeria monocytogenes 264, S. aureus 29/58 (clinical isolate), and C. albicans K2 (clinical strain). The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of dermcidin in its biologically active form.     

Differential Roles of the Two-Component Peptides of Lactocin 705 in Antimicrobial Activity

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705α and Lac705β. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705α/Lac705β peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705β peptide alone. Furthermore, Lac705β increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705α protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Δψ and ΔpH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705α peptide is responsible for receptor recognition, and the Lac705β peptide is the active component on the cell membrane of CRL691 cells. Received: 12 April 2002 / Accepted: 24 May 2002     

Purification and N-terminal amino acid sequence determination of the cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei.

The cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151 was purified to homogeneity by anion-exchange and hydrophobic-interaction chromatography, chromatofocusing and gel-filtration. The purification resulted in a 600-700-fold increase in specific activity of the proteinase and the final yield was approximately 20%. Upon chromatofocusing, two proteolytically active components, termed pro135 and pro110, were detected. pro135 had an isoelectric point of 4.2. It had an Mr of about 300,000 as determined by gel-filtration and 135,000 as judged by SDS-PAGE, indicating that it may exist as a dimer in its native state. pro110 had an isoelectric point of 4.4, and an Mr of about 150,000 as determined by gel-filtration and 110,000 as judged by SDS-PAGE. pro110 appears to be a degradation product of pro135 as they have the same N-terminal amino acid sequence. The first N-terminal amino acid was ambiguous for both components, whereas the sequence from the second to the ninth amino acid was Ala-Lys-Ala-Asn-Ser-Met-Ala-Asn. This is identical to the corresponding sequence of the lactococcal cell-wall-bound proteinases. Although the Lactobacillus proteinase was a little smaller than the lactococcal proteinase, their purification characteristics were very similar, suggesting that these proteinases are related.     

鲈鱼hepcidin原核表达及生物学活性测定

采用RT-PCR方法扩增和克隆了鲈鱼hepcidin(LjFishep)的编码阅读框。该编码阅读框由261个核苷酸组成,编码由86个氨基酸组成的前体蛋白。遗传进化分析表明,LjFishep与条石鲷和河鲈hepcidin的亲缘关系最近。将去除LjFishep信号肽的编码序列克隆到原核表达载体pET-28a(+),实现了LjFishep在大肠杆菌的表达。可溶性分析表明表达蛋白大部分以包涵体形式存在,部分以可溶性形式存在,非变性电泳可见可溶性蛋白存在单体和多聚体组分。镍柱亲和层析法纯化的鲈鱼hepcidin重组表达蛋白(rLjFishep),利用?KTAFPLC(快速蛋白分离纯化系统)进行逐级分离,非变性电泳可见单一rLjFishep可溶性蛋白单体。体外生物学活性分析显示可溶性rLjFishep蛋白单体具有抑制鲈鱼哈维氏弧菌繁殖的能力。这为进一步研究鲈鱼Hepcidin的生物学功能及临床应用奠定了基础。       

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.     

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.     

Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5

A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F–C–K–S–L–P–L–P–L–S–V–K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.     

Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin–SUMO dual fusion system

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli.     

Isolation and biochemical characterization of a novel lantibiotic mutacin from Streptococcus mutans.

Certain members of the indigenous biota of humans produce antimicrobial substances called bacteriocins, which inhibit other bacteria, including members of their own species. One of these substances, mutacin, is made by Streptococcus mutans, a member of the oral biota. Mutacin inhibits other mutans streptococci as well as many gram-positive exogenous pathogens. Here, we report for the first time the purification and partial biochemical characterization of a lanthionine-containing mutacin peptide from S. mutants T8. The biologically active peptide was isolated from the broth cultures by ultrafiltration and differential precipitation. The final mutacin preparation was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. A molecular mass of the peptide was estimated by electrospray ionization mass spectroscopy to be 3,244.64 +/- 1.15 Da. Its amino acid composition indicates the presence of lanthionine and likely beta-methyllanthionine in a total of about 25 amino acids. Because alpha,beta-unsaturated amino acids, the precursors of lanthionine residues, are often found in lantibiotics, we carried out the addition reaction of the mutacin with N-(methyl)mercaptoacetamide. The subsequent electrospray ionization mass spectroscopy analysis indicated the presence of two reaction products with M(r)s of 3,350.45 and 3,456.0. These are interpreted as the mutacin molecule with the addition of one and two molecules of reagent to the unsaturated amino acids, respectively. Sequencing of the peptide revealed an N-terminal amino acid sequence of Asn-Arg-Trp-Trp-Gln-Gly-Val-Val.     

A novel antimicrobial function for a ribosomal peptide from rainbow trout skin

An antimicrobial peptide was purified from skin secretions and epithelial cells of rainbow trout by cation exchange and reversed phase chromatography. Partial N-terminal amino acid sequence of the purified peptide revealed 100% identity with the first 11 residues of a 40S ribosomal peptide from medaka fish. Its molecular mass, determined by matrix-associated laser desorption/ionisation mass spectrometry, was found to be 6676.6Da. These results indicate that this antimicrobial peptide is likely to be the 40S ribosomal protein S30. It is active at submicromolar concentrations, with an effective 50% reduction concentration of 0.02-0.04 microM against Planococcus citreus. Thus, in addition to its conventional function in the cell as part of the small ribosomal subunit, this peptide may play a role in protection against intracellular or extracellular pathogens.     

Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus)

Manganese-superoxide dismutase (Mn-SOD) from Japanese flounder (Paralichthys olivaceus) hepatopancreas has been purified with high purification (781-fold) and recovery (10.8%). The molecular mass of the purified enzyme was estimated to be 26kDa by SDS-PAGE under reducing conditions. In activity staining by native-PAGE, the Japanese flounder Mn-SOD gave three active bands and exhibited KCN-insensitive activity. In addition, the electrophoretic mobility of this enzyme was observed to be faster than that of Japanese flounder Cu,Zn-SOD. On the other hand, the N-terminal amino acid sequence of this Mn-SOD was determined to be 16 amino acid residues, and the sequence showed high homology to other Mn-SODs but not Japanese flounder Cu,Zn-SOD. Analysis of nucleotide and deduced amino acid sequences revealed that the Mn-SOD cDNA consisted of a 64bp 5'-non-coding region, a 675bp open reading frame encoding 225 amino acids, and a 465bp 3'-non-coding region. The first 27 amino acids containing a mitochondria-targeting signal were highly conserved among other Mn-SODs.     

Effect of curing conditions and Lactobacillus casei CRL705 on the hydrolysis of meat proteins

Aims:  The effect of the common curing conditions used during the manufacture of dry fermented sausage on the proteolytic activity of Lactobacillus casei CRL705 against meat proteins was investigated.
Methods and Results:  Hydrolysis of pork muscle sarcoplasmic and myofibrillar proteins was evaluated by SDS-PAGE and reverse phase-HPLC analysis. Ascorbic acid exerted a stimulatory effect on both sarcoplasmic and myofibrillar protein breakdown by Lact. casei CRL705 with the release of hydrophilic peptides and free amino acids, while NaCl and NaNO₂ mainly stimulated myofibrillar degradation.
Conclusions:  Even when processing temperature (25°C) did not positively affect bacterial protein hydrolysis, the presence of curing salts accounted for a remarkable increase in the non-volatile components that constitute taste-active compounds that strongly influence the final flavour of the product.
Significance and Impact of the Study:  To predict the suitability of Lact. casei CRL705 and its proteolytic enzymes as a starter culture for the dry processing of dry fermented sausages.     

Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2–12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.     

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.