Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

Kumar, Adarsh; Tiwari, Sapana; Thavaselvam, Duraipandian; Sathyaseelan, Kannusamy; Prakash, Avinash; Barua, Anita; Arora, Sonia; Rao, Mangala

doi:10.1016/j.pep.2012.04.002

Cited by 9 publications

(8 citation statements)

References 22 publications

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“…For inclusion body washing, non-ionic surfactants are common choices because they are strong enough to separate proteins while weak enough not to harm the protein structure [28]. Especially, Triton X-100 and Triton X-114 are popular non-ionic surfactants known for effectively separating LPS from proteins [23,28-30]. …”

Section: Resultsmentioning

confidence: 99%

Highly purified mussel adhesive protein to secure biosafety for in vivo applications

Choi

Cheong

et al. 2014

Microb Cell Fact

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BackgroundUnique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult.ResultsIn the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151.ConclusionsThrough this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

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Section: Resultsmentioning

confidence: 99%

Highly purified mussel adhesive protein to secure biosafety for in vivo applications

Choi

Cheong

et al. 2014

Microb Cell Fact

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“…The purified rOmp31 and rOmp28 proteins were characterized by Western blot analysis as shown in Fig. 3 and in our previous studies of rOmp28 (9,11).…”

Section: Resultsmentioning

confidence: 99%

“…The omp28 gene of B. melitensis was cloned in pQE30UA in our previous study, and purified rOmp28 protein was prepared as described earlier (9,11). The clone was inoculated into 10 ml LB medium containing kanamycin (25 g/ml) to prepare a starter culture for the expression and purification of the rOmp28 antigen.…”

Section: Methodsmentioning

confidence: 99%

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Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Tiwari

Kumar

Thavaselvam

et al. 2013

Clin Vaccine Immunol

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bBrucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis. Brucellosis is one of the world's major zoonoses, caused by bacteria of the genus Brucella. It is a serious public health problem in India and in other developing countries. The transmission of Brucella infection and its prevalence in a region depend upon several factors, like food habits, methods of processing milk and milk products, social customs, husbandry practices, climatic conditions, socioeconomic status, and environment hygiene (1). The disease has been recognized as one of the most common laboratory-acquired infections; it has been reported to occur in clinical research and production laboratories. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations, and its diagnosis requires microbiological confirmations by means of isolation from blood cultures or demonstration of the presence of specific antibodies by serological tests (2). Blood cultures provide definite proof of brucellosis but may not provi...

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“…Purification was done employing a modified protocol reported earlier [15]. Briefly, 50 ml induced bacterial culture was pelleted and washed twice with 1X Phosphate Buffered Saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis

2017

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BackgroundBrucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay).Method Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23).ResultThe rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student’s t test was performed on the data.ConclusionOmp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.

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Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

Cited by 9 publications

References 22 publications

Highly purified mussel adhesive protein to secure biosafety for in vivo applications

Highly purified mussel adhesive protein to secure biosafety for in vivo applications

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis

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