Cloning and Expression of Antibacterial Goat Lactoferricin from Escherichia coli AD494(DE3)pLysS Expression System

2008 ◽  
Vol 71 (12) ◽  
pp. 2523-2525 ◽  
Author(s):  
GEN-HUNG CHEN ◽  
LI-JUNG YIN ◽  
I-HUA CHIANG ◽  
SHANN-TZONG JIANG
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Propionibacterium Acnes ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
Diffusion Method ◽  
Activity Assay ◽  
E Coli ◽  
Expression Host

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-β-d -thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His·Tag and GLfcin S-His·Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His·Tag and GLfcin S-His·Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His·Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His·Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100°C.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

scholarly journals UJI AKTIVITAS ANTIBAKTERI EKSTRAK DAUN TEMBELEKAN (Lantana camara Linn) TERHADAP PERTUMBUHAN Staphylococcus aureus dan Escherichia coli

Biocelebes ◽  
2022 ◽  
Vol 15 (2) ◽  
pp. 90-97
Author(s):  
Gaby Maulida Nurdin
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Ethanol Extract ◽  
Lantana Camara ◽  
Diffusion Method ◽  
E Coli ◽  
Activity Test ◽  
Growth Inhibitory ◽  
One Way Anova

This study aimed to determine the effect of concentration ethanol extract from tembelakan leaf (Lantana camara Linn)  on bacteria growth of Staphylococcus aureus and Escherichia coli. Extraction was done by maceration using ethanol 96% and then separated using rotary evaporator. Antibacterial activity test of the ethanol extract by Well agar diffusion method. Variation in crude extract saponin used in this study was 5%, 10%, 15%, 20%, 25% and positive controls were used for comparison with Amoxicilin and Chloramphenicole concentration of 25 µg/mL and DMSO as a negative control. The results of antibacterial activity test is indicated by the formation of growth inhibitory region S. aureus and E. coli. The result of growth inhibitory regions was analyzed by One way ANOVA. One way ANOVA test results indicate that there are effects of ethanol extract concentration of tembelekan leaf (L. camara Linn) against S. aureus and E. coli. Effective concentration of ethanol extract tembelekan leaf (L. camara Linn) when compared with positive control to inhibit the growth of S. aureus and E. coli is at 25% with a relatively strong antibacterial activity. Test with phytochemicals screening method which is showed that tembelekan leaf contains the flavanoid, saponins, and tannins compounds as antibacterial

scholarly journals Antibacterial Activity of Liverworts of Dumortiera hirsute (Sw.) Nees Ethyl Acetate Extract Against Pathogenic Bacteria

2021 ◽  
Vol 9 (2) ◽  
pp. 75
Author(s):  
Luthfiah Luthfiah ◽  
Dwi Setyati ◽  
Sattya Arimurti
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Ethyl Acetate ◽  
Pathogenic Bacteria ◽  
Ethyl Acetate Extract ◽  
Antibacterial Properties ◽  
Salmonella Typhi ◽  
Diffusion Method ◽  
E Coli ◽  
Agar Well Diffusion Method

Dumortiera hirsuta is one of the liverworts that can be used as a medicinal to prevent infection by pathogenic bacteria. The content of secondary metabolites of D. hirsuta has potential as antibacterial properties includes flavonoids, alkaloids and steroids. This research is to analyze the antibacterial activity of moss D. hirsuta against pathogenic bacteria that will be beneficial to humans. Liverworts of D. hirsuta were extracted using ethyl acetate solvent and tested against three types of pathogenic bacteria using the agar well-diffusion method. The results of this study indicated that the ethyl acetate extract of D. hirsuta at 100% concentration could inhibit the growth of Escherichia coli, Staphylococcus aureus, and Salmonella typhi bacteria. The range of antibacterial activity categories of the ethyl acetate extract of D. hirsuta to E. coli, S. aureus, and S. typhi between weak to moderate.

scholarly journals ANTIBACTERIAL ACTIVITY OF ROBUSTA COFFEE (COFFEA CANEPHORA L.) LEAVES TO STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI

2019 ◽  
pp. 113-115
Author(s):  
ZAMHARIRA MUSLIM ◽  
YONANIKO DEPHINTO
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Ethyl Acetate ◽  
Antibacterial Effect ◽  
Ethyl Acetate Fraction ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Robusta Coffee

Objective: This research aims to analyze the ability of robusta coffee leaves fraction extract to inhibit the growth of Staphylococcus aureus and Escherichia coli and also determine the minimum inhibitory concentration (MIC). Methods: Antibacterial activity evaluated by the disc diffusion method observed in four types of fraction of extract robusta coffee leaves (n-hexane, ethyl acetate, ethanol, and water). Each extract divided into three various concentrations, 5%, 10%, and 15%. Determination of antimicrobial activity in vitro by the disk diffusion method. Results: Ethyl acetate fraction of coffee leaves extract produced the largest diameter zone of inhibition of bacterial growth compared to other extraction fractions of 17.28 mm in E. coli and 18.58 mm in S. aureus. The MIC of coffee leaves extract fraction water, ethyl acetate, and n-hexane on E. coli and S. aureus is 5%, while the fraction ethanol MIC is 10%. Conclusion: The antibacterial effect of ethyl acetate fraction of coffee leaves extract showed an antibacterial effect that was better than the fraction of n-hexane, ethanol, and water.

Bakteri Simbion Karang Porites dari Perairan Gunungkidul, Yogyakarta dan Aktivitas Antibakteri terhadap Bakteri Patogen Staphylococcus aureus dan Escherichia coli

2018 ◽  
Vol 7 (1) ◽  
pp. 43
Author(s):  
Rivan Novianto Madilana ◽  
Diah Permata Wijayanti ◽  
Agus Sabdono
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
16S Rrna ◽  
Bacillus Pumilus ◽  
Coral Tissue ◽  
Diffusion Method ◽  
Rrna Gene ◽  
E Coli ◽  
Porites Coral

Porites merupakan genus karang yang memiliki persebaran luas di Indonesia, termasuk perairan Gunungkidul, Yogyakarta. Penelitian menunjukkan bahwa bakteri simbion karang Porites memiliki potensi antibakteri dalam menanggulangi bakteri patogen. Penelitian ini betujuan untuk mengetahui jenis bakteri simbion karang Porites dari Perairan Gunungkidul Yogyakarta yang memiliki aktivitas antibakteri patogen S. aureus dan E. coli. Bakteri simbion diisolasi dari fragmen jaringan karang dengan pengenceran bertingkat, kemudian uji aktivitas antibakteri dilakukan dengan menggunakan metode overlay dan difusi paperdisk. Delapan dari 64 isolat aktif menghambat kedua bakteri patogen Staphylococcus aureus dan Escherichia coli. Dua diantaranya merupakan isolat unggul yang menunjukkan aktivitas antibakteri paling baik. Kedua isolat selanjutnya diidentifikasi karakteristik molekular DNA dengan sekuen gen 16S rRNA. Hasil identifikasi 16S rRNA menunjukkan isolat GKP1.4.3 memiliki kesamaan 99% dengan Bacillus pumilus strain NBRC 12092, dan isolat GKP3.2.2 memiliki kesamaan 99% dengan Vibrio natriegens strain NBRC 15636.Porites is a coral which has distributed widely in Indonesia, including Gunungkidul Waters, Yogyakarta. Research has shown that Porites coral symbiont bacteria have antibacterial potency against pathogenic bacteria.This study aims to determine the type of Porites coral symbiont bacteria collected from the waters of Gunungkidul Yogyakarta which has antibacterial activity of S. aureus and E. coli. Bacteria symbionts were isolated from coral tissue fragments by serial dillution method, while antibacterial activity was performed by using overlay and paperdisk diffusion method. Eight of the 64 active isolates inhibited both pathogenic bacteria S. aureus and E. coli. Two of 8 isolates showed stronger antibacterial activity. The two isolates subsequently identified the molecular characteristics of DNA with the 16S rRNA gene sequence. The identification of 16S rRNA showed that GKP1.4.3 isolate had 99% similarity with Bacillus pumilus strain NBRC 12092, and GKP3.2.2 isolate had 99% similarity with Vibrio natriegens strain NBRC 15636.

scholarly journals Antibacterial Activity of Polygonum pulchrum Blume Ethanol Extract on Staphylococcus aureus and Escherichia coli

2018 ◽  
Vol 3 (2) ◽  
pp. 26
Author(s):  
Asman Sadino ◽  
Idin Sahidin ◽  
Wahyuni Wahyuni
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Ethanol Extract ◽  
Inhibition Zone ◽  
Health Concern ◽  
Diffusion Method ◽  
Resistant Bacteria ◽  
E Coli ◽  
Weak Antibacterial Activity

The emergence of resistant bacteria strain has become a global health concern. It encourages the exploration of potential antibacterial agents, particularly from natural sources. The aim of this study was to investigate the antibacterial activity of ethanol extract of root, stems, leaves, and flowers of Polygonum pulchrum Blume against Staphylococcus aureus and Escherichia coli, through disc diffusion method using cup-plate method. Inhibition zone against S. aureus from roots, stems, leaves, and flowers ethanol extract were 3.5 mm, 2.5 mm, 2.25 mm, and 2.62 mm, respectively, while the inhibition zone against E. coli were 2.25 mm, 2.12 mm, 1.62 mm, and 1.75 mm, respectively. In conclusion, ethanol extract of root, stem, leaves, and flower of P. pulchrum Bl possessed weak antibacterial activity against S. aureus and E. coli.Keywords: P. pulchrum Bl, antibacterial, E. coli, S. aureus, cup-plate technique

scholarly journals Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Trang Thi Phuong Phan ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fusion Protein ◽  
Expression System ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

scholarly journals Antibacterial Activity of Sea Cucumber (Stichopus ocellatus) Against Bacillus cereus and Escherichia coli Bacteria

2021 ◽  
Vol 934 (1) ◽  
pp. 012024
Author(s):  
M Sukmiwati ◽  
M Ilza ◽  
A Diharmi ◽  
S Sherrin
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Bacillus Cereus ◽  
Sea Cucumber ◽  
Bioactive Compound ◽  
Parameter Analysis ◽  
Diffusion Method ◽  
Solvent Ratio ◽  
Sea Cucumbers ◽  
E Coli

Abstract Sea cucumber (Stichopus ocellatus) is one of the bioresources that has not been utilized optimally. Samples were collected in the coastal sea of Lengkang Island, Batam. This study was aimed to determine the bioactive compound and antibacterial activity of sea cucumber extract against Bacillus cereus and Escherichia coli. The research method used was an experiment with a series of experiments, namely the extraction of sea cucumbers with ethanol as a solvent. Parameter analysis were identification of bioactive compound (phytochemical) and antibacterial activity against Bacillus cereus and Escherichia coli using well diffusion method. The treatment for the antibacterial activity test used various extract concentrations consisting of 125, 250, 375, and 500 g/mL with three replications. The results of the identification of the bioactive compounds of sea cucumber extract showed that the ethanolic extract of sea cucumbers contained saponins, alkaloids, and phenolics. The analysis of the antibacterial activity of the sea cucumber extract showed that the diameter of the inhibition zone of the ethanol extract at a solvent ratio of 1: 5 (w/v) and at a concentration of 500µ/mL against B. cereus was 14.66 ± 0.37mm and E. coli was 15.45. ±0.17mm. The results showed that the highest antibacterial activity of sea cucumber extract against E. coli bacteria.

scholarly journals Antibacterial Activity of Borassus flabellifer Vinegar-Graphene Quantum Dots Against Gram-Positive and Gram-Negative Bacteria

2021 ◽  
Vol 33 (11) ◽  
pp. 2662-2666
Author(s):  
Amnuay Noypha ◽  
Paweena Porrawatkul ◽  
Nongyao Teppaya ◽  
Parintip Rattanaburi ◽  
Saksit Chanthai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Quantum Dots ◽  
Antibacterial Activity ◽  
Graphene Quantum Dots ◽  
Diffusion Method ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli

Borassus flabellifer vinegar–graphene quantum dots (BFV-GQDs) were successfully synthesized using a pyrolysis method with Borassus flabellifer vinegar (BFV) as the precursor. All the samples were characterized using ultraviolet-visible spectrophotometry (UV-Vis), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The antibacterial activities of BFV-GQDs against strains of Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were determined using the agar well diffusion method for preliminary screening, while minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the broth macro-dilution method. The zones of inhibition were compared with those of citric acid–graphene quantum dots (CA-GQDs). It was observed that the synthesized BFV-GQDs demonstrated excellent antibacterial activity against Staphylococcus aureus (82.3%) and good antibacterial activity against Escherichia coli (73.3%). The MIC of BFV-GQDs against E. coli was 6.25 mg/mL and S. aureus was 12.5 mg/mL, whereas the MBC of BFV-GQDs against E. coli was 12.5 mg/mL and S. aureus was 25.0 mg/mL.

scholarly journals Therapeutic potentials of Excoecaria agallocha against gram-positive and gram-negative fish bacterial pathogens

2019 ◽  
Vol 2 (2) ◽  
pp. 87-94
Author(s):  
Laith A. Abdul Razak ◽  
Nadirah Musa ◽  
Aya Jabar ◽  
Najiah Musa
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Streptococcus Agalactiae ◽  
Oreochromis Niloticus ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Diffusion Method ◽  
Mangrove Plant ◽  
E Coli ◽  
Excoecaria Agallocha

Background: The present investigation of a mangrove plant, Excoecaria agallocha, which is a popular medicinal substitute for the treatment of microbial ailments, were evaluated for potential antimicrobial activity against pathogenic bacteria Escherichia coli and Streptococcus agalactiae in tilapia, Oreochromis niloticus. Methods: Antibacterial activity was performed using agar diffusion method, disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiotic susceptibility assays. Experimental fish fed diet containing 0 (control), 5, 25, and 50 mg kg-1 E. agallocha leaf methanol extract for 28 days then challenged individually with E. coli or S. agalactiae and mortalities were recorded over a ten-day post-infection period.  Results: Results indicated that both bacterial species are sensitive to tetracycline, ampicillin, and amoxicillin. E. coli was found to be resistant to neomycin. E. agallocha extract concentration of 50 mg/ml produced a zone of inhibition of 18 mm against E. coli, in contrast to 13 mm against S. agalactiae.  E. agallocha showed bactericidal activity against E. coli and bacteriostatic activity against S. agalactiae. The highest E. agallocha LC50 activity was 83 mg/ml. The highest cumulative mortality was 90.0 ± 10.0% in control as compared to 26.7 ± 11.5% in the group fed with 50 mg kg-1 E. agallocha extract, significant differences (P < 0.05). Conclusion: Hence, E. agallocha showed antibacterial activity against fish pathogens Escherichia coli and Streptococcus agalactiae in tilapia, Oreochromis niloticus; therefore, E. agallocha may be used as an alternative therapeutic agent against fish pathogenic bacteria as an additive to feed at a concentration depend, safe, non-cytotoxic doses.

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