scholarly journals Construction of prokaryotic expression system of ltB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

2004 ◽  
Vol 10 (18) ◽  
pp. 2675 ◽  
Author(s):  
Jie Yan
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Fusion Gene ◽  
Expression System ◽  
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.

An efficient production of hybrid recombinant protein comprising non-structural proteins (NS 1 & NS 3) of bluetongue virus in prokaryotic expression system

2019 ◽  
Vol 155 ◽  
pp. 15-20 ◽  
Author(s):  
Nihar Nalini Mohanty ◽  
Sathish Bhadravati Shivachandra ◽  
Sanchay Kumar Biswas ◽  
Vijay Nagaraj ◽  
Thaslim Jaglur Basheer ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Bluetongue Virus ◽  
Expression System ◽  
Structural Proteins ◽  
Efficient Production ◽  
Prokaryotic Expression System

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 Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fei Du ◽  
Yun-Qi Liu ◽  
Ying-Shuang Xu ◽  
Zi-Jia Li ◽  
Yu-Zhou Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Rna Polymerase ◽  
Recombinant Proteins ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Expression Level ◽  
Foreign Protein ◽  
Atpase Subunit ◽  
E Coli ◽  
Prokaryotic Expression System

AbstractEscherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

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