Production and characterization of recombinant human Ku antigen.
نویسندگان
چکیده
Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.
منابع مشابه
Production and Characterization of Murine Monoclonal Antibodies to Leishmania Gp63 Antigen
Background : Production of monoclonal antibodies to Leishmania antigens assists the identification and characterization of these organisms. Objective: Production of monoclonal antibodies against epitopes on the gp63. Methods: Two murine monoclonal antibodies to gp63 were produced and characterized. The reactions of both antibodies with soluble leishmanial antigens, purified gp63 and truncated r...
متن کاملFunctional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen
Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore,...
متن کاملProduction and Characterization of Monoclonal Antibodies Recognizing a Common 57-kDa Antigen of Leishmania Species
Background: The therapy of leishmania infection is difficult and each year 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated, therefore, there is a need for an effective vaccine. Monoclonal antibody (mAb) is one of the suitable methods for isolation and purification of leishmania antigens. In this report, we produced several mAb aga...
متن کاملProduction and Characterization of a Monoclonal Antibody against an Antigen on the Surface of Non-Small Cell Carcinoma of the Lung
Background: Lung carcinoma is a multiple type cancer comprising of small cell and non-small cell carcinomas (NSCLC). For therapeutic and diagnostic purposes, serum monoclonal antibodies have been produced against lung cancer. Objective: To charac-terize a murine monoclonal antibody (ME3D11) reactive with human NSCLC. Methods: A murine monoclonal antibody (ME3D11) reactive with human NSCLC was s...
متن کاملCloning, Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections
Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alt...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Nucleic acids research
دوره 22 19 شماره
صفحات -
تاریخ انتشار 1994