scholarly journals Pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues

1996 ◽  
Vol 316 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Iris BRUCHHAUS ◽  
Thomas JACOBS ◽  
Martin DENART ◽  
Egbert TANNICH
Keyword(s):  
Entamoeba Histolytica ◽  
Prokaryotic Expression ◽  
Recombinant Expression ◽  
Expression System ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Prokaryotic Expression System ◽  
Competitive Inhibitors ◽  
Dna Fragment

By using oligonucleotide primers derived from regions highly conserved in prokaryotic and eukaryotic phosphofructokinase sequences, a genomic DNA fragment was amplified and used to isolate cDNA and genomic clones coding for PPi-dependent phosphofructokinase (PPi-PFK) of Entamoeba histolytica. The open reading frame consists of 1308 bp and the corresponding protein has a calculated molecular mass of 47.6 kDa. The N-terminal half of the protein shows 27–35% identity with PPi-PFKs or ATP-dependent phosphofructokinases (ATP-PFKs) of various eukaryotic and prokaryotic organisms. The amino acid residues that form the active site of the PPi-PFK from Propionibacterium freudenreichii and the allosteric ATP-PFK from Escherichia coli are conserved within the amoeba sequence. The PPi-PFK was recombinantly expressed by using a prokaryotic expression system. The purified recombinant protein was found to be enzymically active. The Km values for PPi and fructose 6-phosphate of the native and the recombinant PPi-PFKs were nearly identical. Various bisphosphonates (synthetic pyrophosphate analogues) were tested for their ability to inhibit PPi-PFK activity or amoebic growth. All bisphosphonates tested were competitive inhibitors for amoeba PPi-PFK activity. The best inhibitors were CGP 48048 and zoledronate, with Ki values of 50 μM. All bisphosphonates inhibited amoebic growth. One of them (risedronate) was inhibitory at a concentration of 10 μM. Bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.

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 Chemical modifications of a recombinant bovine stress-inducible 70 kDa heat-shock protein (Hsp70) mimics Hsp70 isoforms from tissues

1995 ◽  
Vol 305 (1) ◽  
pp. 197-203 ◽  
Author(s):  
J A Gutierrez ◽  
V Guerriero
Keyword(s):  
Skeletal Muscle ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Expression System ◽  
Protein A ◽  
Two Dimensional Gel Electrophoresis ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Heat Shock Protein Hsp70 ◽  
Bovine Skeletal Muscle

A cDNA clone for the stress-inducible 70 kDa heat-shock protein (Hsp70) has been isolated from a bovine skeletal-muscle cDNA library. This mRNA encodes a protein with a calculated molecular mass of 70250 Da. The cDNA has one continuous open reading frame capable of encoding a 641-amino-acid protein. Expression of this cDNA in a bacterial expression system produced a protein with a mobility identical with that of the inducible Hsp70 protein from bovine skeletal muscle as determined by SDS/PAGE. Two-dimensional gel electrophoresis demonstrated this protein to have focusing properties identical with that of a minor isoform from bovine skeletal muscle. Upon carbamylation of this bacterially expressed protein, a train of charged proteins with charge differences of -1 were produced. These carbamylated proteins were shown to have similar focusing mobilities to the Hsp70 isoforms isolated from bovine skeletal muscle. These results demonstrate the identification of a skeletal-muscle inducible Hsp70 gene and suggest that the presence of multiple Hsp70 isoforms may be the product of post-translational modifications to the Hsp70 proteins.

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.

Features of the cellodextrinase gene from Fibrobacter succinogenes S85

1994 ◽  
Vol 40 (7) ◽  
pp. 592-596 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Gel Filtration ◽  
Fibrobacter Succinogenes ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Base Pairs ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Promoter Sequences ◽  
Putative Ribosome Binding Site ◽  
Coli Promoter

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.

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.

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