We'd like to understand how you use our websites in order to improve them. Register your interest. Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase MDH , a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein.
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Mycobacterium tuberculosis M. Approximately two billion people worldwide are infected by M. Forty million people are estimated to die because of M. One of the other problems regarding TB is its appropriate diagnosis. The optimization of the cytochrome P CYP protein expression was evaluated in different conditions.
The results of Western Blotting indicated that the purified protein was specifically detected. In this experimental study, for the first time in Iran the expression and purification of this recombinant protein was done successfully. This recombinant protein could be used as a vaccine candidate and diagnostic purpose in subsequent investigations. This successful pathogen has an ability to escape from the immune system by several mechanisms 1 , 2.
TB is an old disease and is regarded as a health problem all over the world, not least in developing countries. Two billion people worldwide are infected by M. On the eve of the 21st century, TB turned into one of the most important health issues across the globe.
Given the existing difficulties regarding the BCG vaccine such as developing disseminated BCG in patients with human immunodeficiency virus HIV and influencing the tuberculin skin test, numerous studies have assessed antigens causing strong cellular immune responses with a view to developing efficacious vaccines against M. On the other hand, the conventional diagnosis of active infection with M. The key aspect in the control of M. The mentioned staining technique does not have the necessary specificity, and sometimes an examination of the clinical sample yields a positive culture test, although the results of microscopic examinations are negative 7 , 8.
There are currently manifold problems concerning the prevention of TB, first and foremost among which are the aforementioned defects in the BCG vaccine and the existing issues relating to the timely diagnosis of this disease. Accordingly, a considerable number of investigations are presently undertaken to detect M.
The recombinant antigens of M. The cytochrome P CYP has been known as a big family of hemoproteins, the proteins containing iron. To date, bacterial P families have been identified and bacteria have been determined to contain the cytochrome P 9 , Because this protein exists in all M. In addition, according to preliminary investigations conducted with appropriate software, this protein, in terms of immunogenicity, contains epitopes which are well represented by the different alleles of major histocompatibility complex classes I and II.
The cytochrome P belongs to the regions of difference 1. Since there are numerous genes in these regions which are not present in the strains of the BCG vaccine and non-tuberculous mycobacterium , these regions have been the focus of a large amount of research Of these genes, the CYP gene has not been yet expressed and the recombinant protein is yet to be assessed in terms of diagnosis and candidate vaccine.
The present experimental study aimed to express and purify the cytochrome P CYP protein to pave the way for further research. A previous study reported the cloning of the cytochrome P CYP gene The optimization of the target protein expression was evaluated in the pET26b-CYP recombinant plasmid with respect to the five quantities below:.
Cell density at induction time optical density at nm wavelength [OD] or OD of 0. Then, to study the expression of the gene in question, a single colony of E. In the next step, the gene expression was confirmed by Western Blotting and then expression was done in a high volume. After cell density registered the OD of interest, several volumes of the medium as uninduced cells were removed and the rest of medium was induced by IPTG at an efficient concentration.
For the lysis and solubilization of the target cells, the cell mass prepared in the previous step was well dissolved in an Erlen containing solution 1 Tris 1M- ethylenediaminetetraacetic acid [EDTA] 0. The obtained solution was transferred to a falcon tube and sonication was done. The upper solution was thereafter discarded, and the obtained precipitation was dissolved in 25cc solution 2 Tris 1M-NaCl- imidazole 2M-Urea Before purification, the solution containing the protein of interest was dialyzed against a chromatography buffer 20 mM Tris, mM Nacl, 5 mM imidazole, 8M Urea.
Dialysis was conducted for two purposes: removing EDTA inside the solution containing the target protein which causes damage to the resin and replacing the chromatography buffer with water. In the next step, the target protein was purified using a column of nickel-nitrilotriacetic acid Ni-NTA. The charged resin was rinsed using 10 mL sterile deionized water and then balanced by 5 mL chromatography buffer. Subsequently, the solution containing the protein of interest was added to a falcon tube containing agarose resin and placed on the shaker at room temperature for 3 - 4 hours.
After the completion of incubation and numerous washings, the solution was transferred to a purification column Qiagen and rinsed with 20 mL chromatography buffer. Afterward, the target protein was removed from the column by the addition of 20 mL elution buffer chromatography buffer containing mM imidazole.
After the investigation of purity, high-purity fractions were collected in a falcon tube and dialyzed against phosphate buffered saline PBS. The experiments showed that E. Therefore, the TBK medium was used for the investigation of gene expression.
The findings indicated that a band in the kDa region relevant to the recombinant cytochrome P CYP protein was obviously represented by different concentrations of IPTG 0. This band was noted for the induced rather than uninduced cultures. The gene in question was well expressed at the initial hours after the addition of IPTG into the culture medium, and over time the rate of expression increased.
The highest expression was obtained by 1 mmol IPTG 16 hours after the initiation of the induction. Finally, the sample with the highest expression was used for the later steps and protein purification Figure 1. Western Blotting has been developed based on the detection of hexahistidine His6 tag in the C-terminal of the target protein and specific binding to the anti-His6-peroxidase antibody.
However, the uninduced culture lacked this band Figure 2. The purification process comprised the binding of the extract containing the recombinant protein of interest to nickel ion-charged nitrilotriacetic NTA resin and washing with an elution buffer.
The protein in question bound to the column through the His6 tag, and the other proteins were rinsed with a chromatography buffer at a low imidazole concentration 20 mmol. Finally, the proteins binding to the His6 tag were removed from the column using a washing buffer containing a high imidazole concentration mmol. The size of this band was consistent with that of the band obtained from the gene expression Figure 3. Column 1: Molecular weight MW standard Fermentas, Cat SM ; Column 2: Uninduced sample; Columns 3 to Lane 1 to 8, eluted protein fractions obtained from washing by chromatography buffer containing mmol imidazole.
To sterilize the purified protein, a 0. TB in conjunction with the agent causing it, M. In TB control, prevention has priority over correct diagnosis. The immunogenicity of a large number of the cellular components of Tubercle bacilli has been studied as a candidate for vaccination against it, and extensive research has been conducted on DNA vaccines, recombinant BCG, and subunit vaccines including recombinant vaccines, among which the production and purification of recombinant proteins is particularly important 1 , For the expression and production of recombinant proteins, the conditions of the experiments should be optimized.
In other words, such different parameters as cell density at the time of induction, post-induction interval, growth temperature, concentration of the inducing substance, and the type of medium contribute fundamentally to the optimization of the expression of recombinant proteins In the present study, the best expression of the recombinant cytochrome P CYP protein was obtained by the TBK medium: initiation of the induction at 0.
The application of these changes in the expression process caused a significant difference in the expression of the target protein.
In the current study, E. Also, E. Relatively easy testing is one of the benefits of the protein expression in E. However, some defects such as a lack of post-translational modification restrict the use of this expression system 21 , In this study, E. One of the important issues concerning the increase in the recombinant protein expression is the use of a vector with promoters with high activity in the host cell.
For example, bacteriophage T7 is a strong and active promoter in E. In this study, the pET26b expression vector was used because it contains bacteriophage T7 and the His6-tag sequence at the C-terminal region. In addition to being highly efficient and compatible with the cloned gene, this vector enjoys acceptable conditions of expression and efficiency and could largely facilitate purification since it contains the His6-tag sequence Generally, the His6-tag sequence has no significant effect on the protein structure and only facilitates the selective binding of the expressed protein to the nickel column The findings of this study support the notion that E.
A significant challenge in biotechnology is the purification of the recombinant protein with a suitable method whose product is a purified protein with structural integrity and biological activity and without any contamination For the purification process, immobilized-metal affinity chromatography was used.
This method is based on protein isolation on the basis of the reaction of electron donor groups at the protein surface e. Then, the protein in question bound to the nickel ion-charged NTA resin and washing was done using an elution buffer.
Because of the above details, the target protein i. The findings of the SDS-PAGE and Western Blotting analyses of the different fractions obtained from protein purification indicated that the His6 sequence of the target protein effectively and completely bound to the column and each one of the fractions contained highly pure protein. The findings of this study are fully consistent with those of different studies previously conducted 16 , 29 on the expression and purification of recombinant proteins in terms of the optimization of the expression and purification of the studied recombinant proteins.
In view of the significance of M. In the present study, the recombinant cytochrome P CYP protein was expressed and purified with high purity and concentration, which should now pave the way for subsequent, complementary studies.
Such studies could investigate the immunogenicity rate of this protein in vitro and in vivo as well as the immunity responses which could contribute to TB diagnosis. This study was funded by grant awarded by the deputy of Research and Technology of Shahrekord University of Medical Sciences.
The author wishes to thank all who cooperated with the study. Mohammad Rabiee Faradonbeh: analyzed the data and wrote the paper. Reza Heidari: prepared the manuscript and did the statistical analysis. Davood Darban-Sarokhalil: collected and coordinated the samples. Financial Disclosure: The authors are students, faculty members, and academic researchers at Shahrekord, Isfahan, Kermanshah, and Mashhad universities of medical sciences. They do not have any conflict of interest.
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Western immunoblot analysis The isolated vesicles and the different sub-cellular extracts see below were subjected to polyacrylamide gel electrophoresis and then blotted onto a PVDF membrane. Proteins were identified using different primary polyclonal antisera at a final dilution of against CdtA, CdtB, CdtC , an anti-Omp50 antiserum at a final dilution of , an anti-HtrA E. For CRP detection,. Anti-rabbit horseradish selleck peroxidase-conjugate was used as a secondary antiserum at a final dilution of , Lipooligosaccharide analysis and staining Lipooligosaccharide LOS samples were prepared from whole-cell lysates 0.
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