کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1173935 1491400 2014 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection
چکیده انگلیسی

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (KD ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 447, 15 February 2014, Pages 64–73
نویسندگان
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