Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.
نویسندگان
چکیده
Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.
منابع مشابه
Cloning, Expression, and Refolding of PPE17 Protein of Mycobacterium Tuberculosis as a Promising Vaccine Candidate
Background: Tuberculosis as a global health problem requires special attention in the contexts of prevention and control. Subunit vaccines are promising strategies to protect burdens of tuberculosis via increasing the BCG protection. The present study aimed to design a vaccine study by means of high-throughput expression and correct refolding of recombinant protein PPE17. Methods: We aimed to c...
متن کاملهمسانهسازی و بیان ژنهای ممزوجی دومن - a1 آنتیژن حفاظتی با سیلوس آنتراسیس و زیرواحد B سم شیگلا در باکتری E. coli
Introduction: Shigella enterotoxin(STxB) is one of the major virulent factors in Shigella dysenteriae type 1 and E. coli O157:H7, which its immunogenicity, adjuvant and delivery characteristic have been proven. Anthrax is a common disease in humans and animals and identifies domain a-1 antibodies of protective antigen(PA) which is 62% of the PA antigens of Bacillus anthracis. Therefore, the pur...
متن کاملIn Silico Prediction of B-Cell and T-Cell Epitopes of Protective Antigen of Bacillus anthracis in Development of Vaccines Against Anthrax
Protective antigen (PA), a subunit of anthrax toxin from Bacillus anthracis, is known as a dominant component in subunit vaccines in protection against anthrax. In order to avoid the side effects of live attenuated and killed organisms, the use of linear neutralizing epitopes of PA is recommended in order to design recombinant vaccines. The present study is aimed at determining the dominant epi...
متن کاملEvaluation of immune response to recombinant Bacillus anthracis LFD1-PA4 chimeric protein
Background: Anthrax is a particularly dangerous infectious disease that affects humans and livestock. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. Domains 4 and 1 of the protective antigen (PA) and lethal factor (LF), respectively, have very high antigenic properties. Aims: In this...
متن کاملAntibody titers of PEG-PLA block copolymer nanosphere containing chimeric recombinant protein of protective antigen and lethal factor of Bacillus anthracis
Introduction: To date, many vaccines have been developed for anthrax but not yet an ideal vaccine. In this study, chimeric protein containing domain 1 lethal factor and domain 4 protective antigens of Bacillus anthracis in copolymer nanocapsules were used to solve the problems caused by existing vaccines and to increase the efficiency of the proposed vaccine. Materials and Methods: In this expe...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Revista Argentina de microbiologia
دوره 48 1 شماره
صفحات -
تاریخ انتشار 2016