scholarly journals Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Xingang Yu ◽  
Auwalu Yusuf Abdullahi ◽  
Sheng Wu ◽  
Weida Pan ◽  
Xianli Shi ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Giardia Lamblia ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Intracellular Localization ◽  
Target Protein ◽  
Antigenic Specificity ◽  
Soluble Form ◽  
Intracellular Location ◽  
Prokaryotic Expression Vector

To study prokaryotic expression and subcellular localization of α-13 giardin in Giardia lamblia trophozoites, α-13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α-13 giardin was determined by fluorescence immunoassay. The results showed that the length of α-13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti-α13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α-13 giardin. Immunofluorescence assays revealed that α-13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α-13 giardin of G. lamblia.

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 Establishing Prokaryotic Expression System of Angiotensin-Converting Enzyme 2 (ACE2) gene in pigs

2020 ◽  
Author(s):  
Hang Xiao ◽  
Xin-Tian Nie ◽  
Xiao-Xia Ji ◽  
Shu-ping Yan ◽  
Bin Zhu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibody ◽  
Prokaryotic Expression ◽  
High Titer ◽  
Expression System ◽  
Angiotensin Converting Enzyme 2 ◽  
Evolution Analysis ◽  
Prokaryotic Expression System ◽  
First Time ◽  
Ace2 Gene

AbstractIn this paper, ACE2 gene of pigs was cloned and the purified protein was obtained via the prokaryotic expression system. Polyclonal antibody of high titer and sensitivity was obtained using Wastar rats immunization method and is then used to determine of the expression of ACE2 using immunohistochemistry. The sequence of ACE2 in pigs covered 2418 nucleotides and coded 805 amino acid (aa) residues. Sequence homology analysis showed that the ACE2 sequence in pigs is highly conserved among species at the nucleotide and amino acid levels. Genetic evolution analysis revealed that ACE2 gene in pigs has the shortest genetic distance with that in goats while residing in a totally different branch from that in zebra fishes. Analysis of protein structure predicted that ACE2 protein is a transmembrane secreted protein with high hydrophilicity, containing a signal peptide sequences locating between 1aa to 17aa. The ACE2 fusion protein expressed (under the induction with 1.0 mmol/L IPTG for 10 h) was of approximately 100 kDa and mainly existed in inclusion body. Wastar rats immunization showed that the titer of the anti-ACE2 antiserum in rats was 1: 3200. Western blot showed that the antibody binds specifically. Immunohistochemistry showed that the ACE2 protein was expressed in all major tissues of pigs. It is the first time that polyclonal antibody of ACE2 in pigs was obtained and the expression of ACE2 was confirmed. These results will provide a basis for investigating on ACE2’s biological activity in pigs.

Polyclonal Antibody Effects on the Human Cardiac 5-HT4(e)Receptors Depend Upon the Expression System

2004 ◽  
Vol 10 (3-4) ◽  
pp. 125-129
Author(s):  
Emmanuella Di Scala ◽  
Stéphanie Rose ◽  
Olivier Hérault ◽  
Jorge Argibay ◽  
Pierre Cosnay ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Expression System ◽  
Antibody Effects

scholarly journals Study of the 49 kDa excretory-secretory protein gene of Trichinella nativa and Trichinella spiralis

2007 ◽  
Vol 44 (3) ◽  
pp. 120-125 ◽  
Author(s):  
B. Zheng ◽  
L. Xiao ◽  
X. Wang ◽  
D. Li ◽  
Y. Lu ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Protein Gene ◽  
Trichinella Spiralis ◽  
Secretory Protein ◽  
Rt Pcr ◽  
Prokaryotic Expression Vector ◽  
Close Relationship ◽  
Bamhi Site ◽  
Relationship Of ◽  
Trichinella Nativa

AbstractTo study the function of the 49 kDa excretory-secretory (ES) protein gene (P49) of Trichinella, the genes was amplified by RT-PCR from RNA of Trichinella spiralis and Trichinella nativa and several Chinese Trichinella isolates of domestic animals, and sequenced after being cloned. The amplified products of these parasites produced bands of about 950 bp. The 97.2 % to 100 % nucleotides identity and 94.3 % to 100 % identity of deduced amino acids among P49 gene of these Trichinella strains showed the close relationship of these parasites. The P49 gene of T. nativa was cloned into the BamHI site of the prokaryotic expression vector pET-30a, and the recombinant vector was expressed. The expressed product was 40.8 kDa in size. In Western blot analysis, the expressed product was reactive to sera of mice infected with T. nativa, T. spiralis and their Chinese geographical strains.

Induction and intracellular localization of the 80-kilodalton heat-shock protein of Neurospora crassa

1992 ◽  
Vol 70 (12) ◽  
pp. 1347-1355 ◽  
Author(s):  
H. S. Roychowdhury ◽  
T. J. MacAlister ◽  
J. W. Costerton ◽  
M. Kapoor
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Neurospora Crassa ◽  
Immunoelectron Microscopy ◽  
Intracellular Localization ◽  
Normal Growth ◽  
Immunogold Labelling ◽  
Subcellular Compartment ◽  
Intracellular Location ◽  
Gold Label

The most abundant heat-shock protein of Neurospora crassa is a multimeric glycoprotein of 80-kilodaltons (i.e., HSP80), induced strongly by hyperthermia and at a lower level by sodium arsenite, ethanol, and carbon source depletion. Immunoelectron microscopy, using indirect immunogold labelling demonstrated that HSP80 was undetectable in mycelium cultured at the normal growth temperature of 28 °C, but it appeared rapidly following the commencement of heat-shock treatment at 48 °C. HSP80, visualized by the gold label, was observed almost exclusively in the cytoplasm, exhibiting a uniform distribution. Association of this protein with cellular membranes and (or) targeting to a particular subcellular compartment or organelle was not apparent.Key words: 80-kilodalton heat-shock protein, Neurospora, intracellular location, immunoelectron microscopy.

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
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