Selective Isolation of Myosin Subfragment-1 with a DNA-Polyoxovanadate Bioconjugate

Abstract

The bioconjugation of a polyoxometalate (POMs), i.e., dodecavanadate (VO), to DNA strands produces a functional labeled DNA primer, VO-DNA. The grafting of DNA primer onto streptavidin-coated magnetic nanoparticles (SVM) produces a novel composite, VO-DNA@SVM. The high binding-affinity of VO with the ATP binding site in myosin subfragment-1 (S1) facilitates favorable adsorption of myosin, with an efficiency of 99.4% when processing 0.1 mL myosin solution (100 μg mL) using 0.1 mg composite. Myosin adsorption fits the Langmuir model, corresponding to a theoretical adsorption capacity of 613.5 mg g. The retained myosin is readily recovered by 1% SDS (m/m), giving rise to a recovery of 58.7%. No conformational change is observed for myosin after eliminating SDS by ultrafiltration. For practical use, high-purity myosin S1 is obtained by separation of myosin from the rough protein extract from porcine left ventricle, followed by digestion with α-chymotryptic and further isolation of S1 subfragment. The purified myosin S1 is identified with matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry, giving rise to a sequence coverage of 38%.

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells

“…After the initial incubation time, the samples were subjected to UV irradiation for varying quantities of time, and SDS–PAGE analysis was carried out using similar techniques as described above to determine the extent of covalent labeling (Figure a,b). Under these conditions, 50% labeling was observed within 10 min of UV activation (Figure b), which is comparable to previously reported photoaffinity labeling strategies. , …”

“…Under these conditions, 50% labeling was observed within 10 min of UV activation (Figure 3b), which is comparable to previously reported photoaffinity labeling strategies. 39,40 Although the MG probe only becomes fluorescent after binding to the FAP, we were still curious to know whether the diazirine could react nonspecifically with other proteins. To investigate this question, we carried out the labeling reaction in a whole-cell lysate both with and without the addition of FAP (Figure 4).…”

State of the art on the separation and purification of proteins by magnetic nanoparticles

“…For myosins, Chen and colleagues [ 21 ] purposed to purify myosin subfragment-1 using a synthesized DNA-polyoxovanadate biocomposite attached to streptavidin-coated MNPs (V12O32-DNA@SVM) and observed that as little as 0.1 mg of the nanocomposite could efficiently absorb the myosin subfragment-1 based on the intensive binding at the ATP-V12O32 junction. This purification technique had an efficiency rate of 99.4% and serves as the backdrop for further application in myosin extraction from porcine crude protein.…”