scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI

2020 ◽  
Vol 2 (338) ◽  
pp. 20-30
Author(s):  
E. T. Tailakova ◽  
S. О. Sadikaliyeva ◽  
G. O. Shynybekova ◽  
A. K. Abubakirova ◽  
K. T. Sultankulova ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Target Genes ◽  
Guanidine Hydrochloride ◽  
Public Health Problem ◽  
Sodium Deoxycholate ◽  
Expression And Purification ◽  
Important Public Health Problem ◽  
E Coli ◽  
Wash Buffer ◽  
Cellular Immune

Brucellosis is still an important public health problem as long as natural reservoirs of infection exist. Currently, live attenuated vaccines based on strains S19, RB51 and Rev1 are used for the prevention of brucellosis in animals, the main disadvantage of which is virulence for humans. However, animal immunization programs should be implemented to reduce the incidence of humans. The development of safe and effective new generation vaccines using “omix” technology is a promising direction of vaccinology. A number of immunogenic Brucella proteins that elicit both a humoral and cellular immune response has been identified. The aim of these research was to optimize the expression and purification conditions of the Brucella spp. recombinant proteins L7/L12 and SodC. As a result, expressing plasmids pET/Br-L7/L12 and pET/Br-SodC were obtained. The parameters of target genes expression in E. coli were established and the method for purification of recombinant proteins was optimized. Purification of the L7/L12 protein was performed under hybrid conditions on HisPur agarose using a binding buffer containing 6 M guanidine hydrochloride, a wash buffer with 20 mM imidazole and an elution buffer with 300 mM imidazole. Protein SodC was purified under denaturing conditions with the addition of 1 % Triton X-100 and 1 % sodium deoxycholate to the lysis buffer. Inclusions were solubilized with a buffer containing 8 M urea and 5 mM imidazole. The target protein was eluted from HisPur agarose with buffer containing 8 M urea and 100 mM imidazole. The use of modified purification protocols made it possible to obtain purified recombinant proteins with a yield of 13 mg/L for the L7/L12 protein and 10 mg/L for the protein SodC, respectively. The specificity of the proteins was confirmed by a Western blot. Immunization of mice with recombinant proteins led to the production of specific antibodies, the titer of which in ELISA was 1:20480 and 1:20480, respectively.

Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 203-208 ◽  
Author(s):  
Hongmei Dong ◽  
Xiaohu Xu ◽  
Mohong Deng ◽  
Xiaojun Yu ◽  
Hu Zhao ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Target Genes ◽  
Review Process ◽  
Peer Review Process ◽  
Nitrilotriacetic Acid ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Expression Plasmids ◽  
Selection Of

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28–349 aa), MAC2 (166–369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni–NTA, were ~80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28–349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.

scholarly journals Antimicrobial Resistance of DiarrheagenicEscherichia coli Isolated from Children under the Age of 5 Years from Ifakara, Tanzania

1999 ◽  
Vol 43 (12) ◽  
pp. 3022-3024 ◽  
Author(s):  
Jordi Vila ◽  
Martha Vargas ◽  
Climent Casals ◽  
Honorato Urassa ◽  
Hassan Mshinda ◽  
...  
Keyword(s):  
Developing Countries ◽  
Public Health Problem ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Important Public Health Problem ◽  
Important Public Health ◽  
E Coli ◽  
High Level ◽  
Level Resistance ◽  
Children Under 5

ABSTRACT Diarrhea caused by multidrug-resistant bacteria is an important public health problem among children in developing countries. The prevalence and antimicrobial susceptibility of diarrheagenicEscherichia coli in 346 children under 5 years of age in Ifakara, Tanzania, were studied. Thirty-eight percent of the cases of diarrhea were due to multiresistant enterotoxigenic E. coli, enteroaggregative E. coli, or enteropathogenicE. coli. Strains of all three E. colicategories showed high-level resistance to ampicillin, tetracycline, co-trimoxazole, and chloramphenicol but were highly susceptible to quinolones. Guidelines for appropriate use of antibiotics in developing countries need updating.

scholarly journals Expression and purification of four truncated 56 kDa antigens from Orientia tsutsugamushi in Escherichia coli

2018 ◽  
Vol 16 (3) ◽  
pp. 533-541
Author(s):  
Le Thi Lan Anh ◽  
Trinh Van Toan ◽  
Pham Thi Ha Giang ◽  
Bui Thi Thanh Nga ◽  
Vo Viet Cuong ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Affinity Column ◽  
Orientia Tsutsugamushi ◽  
Expression And Purification ◽  
E Coli ◽  
Clinical Presentations ◽  
Insoluble Form

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to humans by the bite of the larva of trombiculid mites. Diagnosis of scrub typhus is normally based on the clinical presentations. However, it is difficult to differentiate scrub typhus from other acute febrile illnesses, such as dengue fever, malaria and leptospirosis due to similar symptoms. For differential diagnosis of scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. In order to produce an ELISA kit for detection of antibodies against O. tsutsugamushi in Vietnam, four truncated 56 kDa antigenic genes of O. tsutsugamushi including Karp (HT-09), Gilliam (HT-11), TA763 (HT-49), and Kato (YB-50) íolated from the most prevalent cases in Vietnam were cloned and expressed in E. coli Rosetta 1 cells. The recombinant proteins formed inclusion bodies when expressed in E. coli. The recombinant 56 kDa proteins in insoluble form were solubilized in 6M urea and were successfully purified by Ni2+affinity column. The purity of four recombinant proteins,HT-09, HT-11, HT-49 and YB-50,reached more than 95% and their concentrations are 12,57 mg/ml; 11,6 mg/ml; 8,98 mg/ml và 8,02 mg/ml, respectively.

scholarly journals Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jitendra Singh Rathore ◽  
Lalit Kumar Gautam
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Recombinant Proteins ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Xenorhabdus Nematophila ◽  
Expression And Purification ◽  
Toxicity Assay ◽  
Neutralization Activity ◽  
E Coli

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

Mineral water: a microbiological approach

2012 ◽  
Vol 12 (5) ◽  
pp. 556-562 ◽  
Author(s):  
Maria Fernanda Falcone-Dias ◽  
Guilherme L. Emerick ◽  
Adalberto Farache-Filho
Keyword(s):  
Mineral Water ◽  
Microbiological Quality ◽  
Public Health Problem ◽  
Quality Standard ◽  
Plate Count ◽  
Important Public Health Problem ◽  
Total Coliforms ◽  
E Coli ◽  
Bottled Mineral Water

The microbiological quality of bottled mineral water of various domestic brands sold in Brazil was investigated, with particular focus on the heterotrophic plate count (HPC). Neither total coliforms nor Escherichia coli were found in any 1.5 L bottle samples. Total coliforms were found in 2.9% of the small bottles, while in 20 L bottles the presence of total coliforms and E. coli was demonstrated in 15.5 and 2.4% of samples, respectively. Pseudomonas aeruginosa was detected in 4.3, 4.5 and 9.5% of small, 1.5 and 20 L bottles, respectively. In 36.4% of the samples of 1.5 L bottles, the HPC was above 500 cfu/mL. This percentage of samples with an HPC above 500 cfu/mL increased to 52.0 and 61.9% in small and 20 L bottles, respectively. Higher contamination by total coliforms, E. coli, P. aeruginosa and HPCs occurred in 20 L bottles. In conclusion, several samples in this study were outside the international quality standard for mineral water and the large number of samples with high HPCs shows that more work must be done on the use of HPC in mineral water and the damaging effects that these microorganisms may cause to humans. The bottled mineral water was confirmed as a particularly important public health problem, due to the poor microbiological quality of the products that are marketed.

scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI

2020 ◽  
Vol 2 (338) ◽  
pp. 20-30
Author(s):  
E. T. Tailakova ◽  
◽  
S. О. Sadikaliyeva ◽  
G. O. Shynybekova ◽  
A. K. Abubakirova ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Expression And Purification ◽  
E Coli

scholarly journals Engineering for an HPV 9-valent Vaccine using Genomic Constitutive Over-expression and Low Lipopolysaccharide Levels in Escherichia Coli Cells

2021 ◽  
Author(s):  
Kaihang Wang ◽  
Lizhi Zhou ◽  
Tingting Chen ◽  
Qiong Li ◽  
Jiajia Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Target Genes ◽  
Plasmid Vector ◽  
Genetically Engineered ◽  
Expression Cassette ◽  
E Coli ◽  
Recombinant Strains ◽  
Endotoxin Contamination

Abstract BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD­—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. ConclusionReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

scholarly journals Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kaihang Wang ◽  
Lizhi Zhou ◽  
Tingting Chen ◽  
Qiong Li ◽  
Jiajia Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Target Genes ◽  
Vaccine Candidate ◽  
Genetically Engineered ◽  
Expression Cassette ◽  
E Coli ◽  
Recombinant Strains ◽  
Endotoxin Contamination

Abstract Background The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. Results Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. Conclusions Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Suicide on the Indian Subcontinent

Crisis ◽  
2002 ◽  
Vol 23 (3) ◽  
pp. 104-107 ◽  
Author(s):  
Murad M. Khan
Keyword(s):  
Sri Lanka ◽  
Mental Health Professionals ◽  
Indian Subcontinent ◽  
Interpersonal Relationship ◽  
Public Health Problem ◽  
Elderly Subjects ◽  
Important Public Health Problem ◽  
Policy Makers ◽  
Formidable Challenge ◽  
Causative Factors

Summary: The Indian subcontinent comprises eight countries (India, Pakistan, Bangladesh, Nepal, Sri Lanka, Afghanistan, Bhutan, and the Maldives) and a collective population of more than 1.3 billion people. 10% of the world's suicides (more than 100,000 people) take place in just three of these countries, viz. India, Sri Lanka, and Pakistan. There is very little information on suicides from the other four countries. Some differences from suicides in Western countries include the high use of organophosphate insecticides, larger numbers of married women, fewer elderly subjects, and interpersonal relationship problems and life events as important causative factors. There is need for more and better information regarding suicide in the countries of the Indian subcontinent. In particular, studies must address culture-specific risk factors associated with suicide in these countries. The prevention of this important public health problem in an area of the world with myriad socio-economic problems, meager resources, and stigmatization of mental illness poses a formidable challenge to mental health professionals, policy makers, and governments of these countries.

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