scholarly journals High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System inEscherichia coli

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Yong-Gang Xie ◽  
Fei-Fei Han ◽  
Chao Luan ◽  
Hai-Wen Zhang ◽  
Jie Feng ◽  
...  

OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1 l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity againstE. coliK88. The intein system is the best currently available system for the cost-effective production of OG2.

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.


1999 ◽  
Vol 6 (4) ◽  
pp. 332-335 ◽  
Author(s):  
Jennifer A Crocket ◽  
Eric YL Wong ◽  
Dale C Lien ◽  
Khanh Gia Nguyen ◽  
Michelle R Chaput ◽  
...  

OBJECTIVE: To evaluate the yield and cost effectiveness of transbronchial needle aspiration (TBNA) in the assessment of mediastinal and/or hilar lymphadenopathy.DESIGN: Retrospective study.SETTING: A university hospital.POPULATION STUDIED: Ninety-six patients referred for bronchoscopy with computed tomographic evidence of significant mediastinal or hilar adenopathy.RESULTS: Ninety-nine patient records were reviewed. Three patients had two separate bronchoscopy procedures. TBNA was positive in 42 patients (44%) and negative in 54 patients. Of the 42 patients with a positive aspirate, 40 had malignant cytology and two had cells consistent with benign disease. The positive TBNA result altered management in 22 of 40 patients with malignant disease and one of two patients with benign disease, thereby avoiding further diagnostic procedures. The cost of these subsequent procedures was estimated at $27,335. No complications related to TBNA were documented.CONCLUSIONS: TBNA is a high-yield, safe and cost effective procedure for the diagnosis and staging of bronchogenic cancer.


2013 ◽  
Vol 3 ◽  
pp. 117-122 ◽  
Author(s):  
TB Ghimire ◽  
NS Thakur

Area and production of raw jute has decreased, though there is a high demand of raw jute in the country. In order to assess production constraints, a survey was carried out in 2005/06 in Jhapa, Morang and Sunsari districts. The study revealed that unstable or low price of raw jute, unavailability of quality jute seed, limited irrigation water at sowing period, diseases complex (wilt), labor shortage during peak season, weed problem, lack of retting water/retting pond were the main constraints in jute production and processing. The study indicates that the maximum production cost has involved in fiber extraction (16.9%) and weeding (16.33%). Jute productivity ranged from 1788 to 2260 kg per hectare. JRO-524 variety of jute has been widely grown across the region due to its wider adaptability, high yield potential and quality fiber. Jute area has been replaced by sugarcane due to its high yield potential and high profit margin. It is observed that the cost of production of jute is high as compared to other crops in the season. Average cost of production of fiber was estimated to be Rs.1563/quintal. For the promotion of jute cultivation in the eastern Terai, it would be better to provide subsidies on seeds and fertilizer to jute growers as practiced in neighboring countries thereby profit margin becomes high and will encourage growers in producing more raw jute within the country for the fulfillment of raw jute requirement of local jute industries. Cost effective technologies have to be developed in jute production and processing aspects for lowering the production cost and increasing the profit margin. Popular genotypes JRO- 524 which was widely adopted needs to be recommended officially for the general cultivation in this region. Being an eco-friendly crop, promotion is required to adapt climate change effect and maintaining the soil properties in jute growing areas. Agronomy Journal of Nepal (Agron JN) Vol. 3. 2013, Page 117-122 DOI: http://dx.doi.org/10.3126/ajn.v3i0.9013


Author(s):  
С. В. Маслійов ◽  
О. О. Беседа ◽  
В. Ф. Дрель ◽  
В. О. Арсієнко

Важко сьогодні уявити технологію вирощування зернових культур без використання регуляторів росту. І це зрозуміло. Адже, ведучи мову про рентабельне виробництво зерна, ми неодмінно асоціюємо це з підвищенням рівня врожайності, яке неможливе без зростання індивідуальної продуктивності рослин. Застосування цих препаратів у поєднанні зі збільшенням культури землеробства та збільшенням масштабів застосування добрив сприяло значному росту урожайності озимої пшениці не тільки в дослідах, але й на виробничих масивах. Однак аналіз рівня урожаїв, які були отримані в Луганській області в наступні роки, показав, що величина його не завжди гарантується. Особливо різке падіння урожаїв в окремі роки здавалось незрозумілим та неприйнятним після запровадження у виробництво прийомів, заснованих на застосуванні регуляторів росту. Аналіз причин різкого падіння урожаїв озимої пшениці в окремі роки показав, що воно зумовлено, перш за все, епіфітотіями – грибковими захворюваннями та ушкодженням рослинними шкідниками. Особливо різке зниження урожаїв відбувається в роки, коли паралельно з’являються два або три з перелічених факторів. Вчені дійшли висновку, що після впровадження у виробництво високоврожайних сортів, застосування ретардантів росту потрібно проводити й боротьбу з хворобами та шкідниками на посівах озимої пшениці. Адже тільки на посівах, захищених від цих факторів, є можливість отримати високопродуктивні урожаї. Nowadays it is difficult to imagine the technology of cultivating grain crops without the use of grow regulators. And this is understandable. After all, speaking about the cost-effective production of grain, we necessarily associate this with an increase in the level of yield, which is impossible without the growth of individual productivity of plants. The use of these drugs in conjunction with the increase in the culture of agriculture and the increase in the use of fertilizers contributed to a significant increase in winter wheat yields not only in experiments, but also in production massifs. However, analysis of the level of yields that was obtained in Luhansk region in next years has shown that its size is not always guaranteed. Especially sharp drop in yields in some years seemed incomprehensible and unacceptable after introducing into the production of techniques based on the use of growth regulators. Analysis of the causes of a sharp drop in winter wheat yields in some years has shown that it is due primarily to the manifestation of epiphytotic diseases – fungal diseases and damage to plant pests. Especially sharp decrease of crops occurs in years when two or almost three of the listed factors appear simultaneously. Scientists came to the conclusion that after introduction of the application of growth retardants into production of high-yielding varieties, it is necessary to carry out the fight against diseases and pests on winter wheat crops. After all, only crops protected from these factors have the opportunity to obtain high yield crops.


2015 ◽  
Vol 60 (1) ◽  
pp. 495-506 ◽  
Author(s):  
Jong-kook Lee ◽  
Chang Ho Seo ◽  
Tudor Luchian ◽  
Yoonkyung Park

ABSTRACTCA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits cytotoxicity toward mammalian cells. Our objective was to identify CA-MA analogues with reduced cytotoxicity by systematic replacement of amino acids with positively charged R groups (His and Lys), aliphatic R groups (Leu), or polar R groups (Glu). Among the CA-MA analogues studied (CMA1 to -6), CMA3 showed the strongest antimicrobial activity, including against drug-resistantEscherichia coliandPseudomonas aeruginosastrains isolated from hospital patients. CMA3 appeared to act by inducing pore formation (toroidal model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little cytotoxicity toward human red blood cells (hRBCs) or HaCaT cells. Additionally, no fluorescence was released from small or giant unilamellar vesicles exposed to 60 μM CMA3 for 80 s, whereas fluorescence was released within 35 s upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide (LPS)-neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS after infection byEscherichia colishowed improved survival after administration of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a mouse model of septic shock, CMA3 reduced the levels of proinflammatory factors, including both nitric oxide and white blood cells, and correspondingly reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/antiendotoxin peptide that could serve as the basis for the development of anti-inflammatory and/or antimicrobial agents with low cytotoxicity.


2018 ◽  
Vol 56 (2A) ◽  
pp. 89-98
Author(s):  
Tran Thi Bich Quyen

A green and simple approach has been successfully developed to synthesize chitosan/Ag nanocomposites using kumquat extract as a biological reducing agent. It indicates to be an eco-friendly and green method for the synthesis providing a cost effective and an efficient route for the chitosan/Ag nanocomposites’ synthesis. The prepared chitosan/Ag nanocomposites have been characterized by UV-vis, TEM, FTIR, and XRD. Result showed those chitosan/Ag nanocomposites have been obtained with average particle size ~15-25 nm. Moreover, the synthesized chitosan/Ag nanocomposites also showed their efficient antimicrobial activity against S. aureus and E. coli. The chitosan/Ag nanocomposite was found to have significantly higher antimicrobial activity than its components at their respective concentrations. The presence of a small percentage (2.5 %, w/w) of metal nanoparticles in the nanocomposite was enough to significantly enhance inactivation of S. aureus and E. coli as compared with unaltered chitosan. Thus, this eco-friendly method could be a competitive alternative to the conventional physical/chemical methods used for the synthesis of chitosan/Ag nanocomposites. Since, it has a potential to use in biomedical and cosmetic applications. 


2020 ◽  
Vol 18 (3) ◽  
pp. 233-238
Author(s):  
Rodríguez-Alejandro C.I. ◽  
M.C. Gutiérrez

Introduction: Currently animal venoms are considered a potential source of numerous bioactive peptides of biochemical and pharmacological interest, such as peptides with antithrombotic, anticoagulant and antimicrobial activity. Methods: Such is the case of the venom from the genus Scolopendromorpha, where numerous PAMs ranging from 2.5 to 4.4 kDa have been purified, they are broad spectrum isolates only of S. subspinipes mutilans. Results: In this study, an antimicrobial peptide (SPC13) of 13 kDa, present in the venom of Scolopendra polymorpha was purified by electroelution and presented antimicrobial activity against S. aureus and P. aeruginosa with MIC of 45 and 192.5 μg/ml respectively, as well as bacteriostatic activity against E. coli at a concentration of 155μg/ml. Conclusion: Additionally, this peptide has a 20.5% hemolytic activity. A partial sequence of SPC13 showed 98% identity with the histone H3 reported in S. viridis (GenkBank: DQ222181.1).


Insects ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 676 ◽  
Author(s):  
Joon Ha Lee ◽  
Hoyong Chung ◽  
Yong Pyo Shin ◽  
In-Woo Kim ◽  
Sathishkumar Natarajan ◽  
...  

Antimicrobial peptides (AMPs) are the frontline innate defense system evolutionarily preserved in insects to combat invading pathogens. These AMPs could serve as an alternative to classical antibiotics to overcome the burden of treating multidrug resistant bacteria. Psacotheasin, a knottin type AMP was isolated from Psacothea hilaris and shown to exhibit antimicrobial activity, especially against fungi through apoptosis mediated cell death. In this study, we aimed to identify novel probable AMPs from Psacothea hilaris, the yellow spotted longicorn beetle. The beetle was immunized with the two bacterial strains (E. coli and S. aureus), and the yeast strain C. albicans. After immunization, total RNA was isolated and sequenced in Illumina platform. Then, beetle transcriptome was de novo assembled and searched for putative AMPs with the known physiochemical features of the AMPs. A selection of AMP candidates were synthesized and tested for antimicrobial activity. Four peptides showed stronger activity against E. coli than the control AMP, melittin while one peptide showed similar activity against S. aureus. Moreover, four peptides and two peptides showed antifungal activity stronger than and similar to melittin, respectively. Collectively one peptide showed both antibacterial and antifungal activity superior to melittin; thus, it provides a potent antimicrobial peptide. All the peptides showed no hemolysis in all the tested concentrations. These results suggest that in silico mining of insects’ transcriptome could be a promising tool to obtain and optimize novel AMPs for human needs.


2019 ◽  
Author(s):  
Meron R Ghidey ◽  
S M Ashiqul Islam ◽  
Grace Pruett ◽  
Christopher Michel Kearney

AbstractAs antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants compared toE. coliexpression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. We wondered if there were a physical factor that made heterologous AMPs difficult to express in plants. Using a meta-analysis of protein databases, we determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. To apply this finding to plant expression, we tested the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to −5, in the the tobacco,Nicotiana benthamiana. We first tested several carrier proteins and were able to express AMPs only with elastin-like polypeptide (ELP). Conveniently, ELP fusion allows for a simple, cost-effective temperature shift purification. Using the ELP system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity againstStaphylococcus epidermidiswas an order of magnitude stronger (average MIC = 0.26 µM) than that typically seen for AMPs expressed inE. coliexpression systems. Unexpectedly, this high level of antimicrobial activity was associated with the uncleaved fusion peptide. In contrast, all previous reports of AMPs expressed in both plant andE. coliexpression systems show cleavage from the fusion partner to be required before activity is seen. In summary, we describe a means of expressing AMP fusions in plants in high yield, purified with a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity, without the need for a peptide cleavage step.


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