scholarly journals Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

2009 ◽  
Vol 75 (12) ◽  
pp. 3980-3986 ◽  
Author(s):  
Su A Jang ◽  
Bong Hyun Sung ◽  
Ju Hyun Cho ◽  
Sun Chang Kim
Keyword(s):  
Antimicrobial Peptides ◽  
Prokaryotic Expression ◽  
Inclusion Bodies ◽  
Expression System ◽  
Cost Effective ◽  
Exchange Chromatography ◽  
Insoluble Complex ◽  
Prokaryotic Expression System ◽  
Natural Forms ◽  
Cleavage Step

ABSTRACT We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of Escherichia coli culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.

Inside Cover: Implantation of Post-translational Tyrosylprotein Sulfation into a Prokaryotic Expression System (ChemBioChem 3/2011)

ChemBioChem ◽  
2011 ◽  
Vol 12 (3) ◽  
pp. 338-338
Author(s):  
Lu-Yi Lu ◽  
Bo-Han Chen ◽  
Jennifer Yun-Shin Wu ◽  
Chen-Chu Wang ◽  
Da-Huang Chen ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Expression System ◽  
Prokaryotic Expression System

scholarly journals Vaccine Efficacy of Bm86 Ortholog ofH. a. anatolicum, rHaa86 Expressed in Prokaryotic Expression System

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
P. Azhahianambi ◽  
D. D. Ray ◽  
Pallab Chaudhuri ◽  
Rohita Gupta ◽  
Srikanta Ghosh
Keyword(s):  
Vaccine Efficacy ◽  
Prokaryotic Expression ◽  
Integrated Control ◽  
Expression System ◽  
Fusion Tag ◽  
E Coli ◽  
Adult Females ◽  
Prokaryotic Expression System ◽  
Integrated Tick Management ◽  
Tick Vaccine

The use of tick vaccine in controlling ticks and tick borne diseases has been proved effective in integrated tick management format. For the control ofH. a. anatolicum, Bm86 ortholog ofH. a. anatolicumwas cloned and expressed as fusion protein inE. coliasE. coli-pETHaa86. The molecular weight of the rHaa86 was 97 kDa with a 19 kDa fusion tag of thioredoxin protein. The expressed protein was characterized immunologically and vaccine efficacy was evaluated. After 120 hours of challenge, only 26% tick could successfully fed on immunized animals. Besides significant reduction in feeding percentages, a significant reduction of 49.6 mg;P<.01in the weight of fed females in comparison to the females fed on control animals was recorded. Following oviposition, a significant reduction of 68.1 mg;P<.05in the egg masses of ticks fed on immunized animals in comparison to the ticks fed on control animals was noted. The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 73.8%, 31.3%, 15.8%, and 82.3%, respectively. The results indicated the possibility of development of rHaa86 based vaccine as a component of integrated control of tick species.

scholarly journals Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA

2009 ◽  
Vol 8 (3) ◽  
pp. 1147-1157 ◽  
Author(s):  
J.B. Cajazeiras ◽  
L.M. Melo ◽  
E.S. Albuquerque ◽  
G. Rdis-Baptista ◽  
B.S. Cavada ◽  
...  
Keyword(s):  
Protein Expression ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Capra Hircus ◽  
Prokaryotic Expression System

scholarly journals Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

2011 ◽  
Vol 5 (12) ◽  
pp. 856-862 ◽  
Author(s):  
Fakhri Haghi ◽  
Shahin Najar Peerayeh ◽  
Seyed Davar Siadat ◽  
Mehran Montajabiniat
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Prokaryotic Expression ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Expression System ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Prokaryotic Expression Vector

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

scholarly journals Functional Analysis of Peptidyl-prolyl cis-trans Isomerase from Aspergillus flavus

2019 ◽  
Vol 20 (9) ◽  
pp. 2206 ◽  
Author(s):  
Saleem Ahmad ◽  
Sen Wang ◽  
Weizhong Wu ◽  
Kunlong Yang ◽  
YanFeng Zhang ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Prokaryotic Expression ◽  
Cell Cycle Control ◽  
Expression System ◽  
Homology Model ◽  
Wild Type ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Prokaryotic Expression System ◽  
Type Strains

Aspergillus flavus, a ubiquitous filamentous fungus found in soil, plants and other substrates has been reported not only as a pathogen for plants, but also a carcinogen producing fungus for human. Peptidyl-Prolyl Isomerase (PPIases) plays an important role in cell process such as protein secretion cell cycle control and RNA processing. However, the function of PPIase has not yet been identified in A. flavus. In this study, the PPIases gene from A. flavus named ppci1 was cloned into expression vector and the protein was expressed in prokaryotic expression system. Activity of recombinant ppci1 protein was particularly inhibited by FK506, CsA and rapamycin. 3D-Homology model of ppci1 has been constructed with the template, based on 59.7% amino acid similarity. The homologous recombination method was used to construct the single ppci1 gene deletion strain Δppci1. We found that, the ppci1 gene plays important roles in A. flavus growth, conidiation, and sclerotia formation, all of which showed reduction in Δppci1 and increased in conidiation compared with the wild-type and complementary strains in A. flavus. Furthermore, aflatoxin and peanut seeds infection assays indicated that ppci1 contributes to virulence of A. flavus. Furthermore, we evaluated the effect of PPIase inhibitors on A. flavus growth, whereby these were used to treat wild-type strains. We found that the growths were inhibited under every inhibitor. All, these results may provide valuable information for designing inhibitors in the controlling infections of A. flavus.

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