Bacterial Production of Recombinant Coagulation Factor VIII Domains.

Factor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this study, we generated Glutathione S-transferase (GST)-conjugated recombinant A2 and A3 domains of F8 using Escherichia coli. The high growth rate and economically advantageous protein production system in terms of inexpensive reagents and materials in E. coli cells facilitated the completion of entire process from protein expression to purification in 3-4 days with low production cost. Subsequent assessment of these purified proteins using enzyme-linked immunosorbent assay (ELISA) and antibodies against F8 revealed enhanced detection of rF8-A2 or rF8-A3 in a concentration dependent manner, indicating the presence of the antibody-binding epitopes in these proteins. Furthermore, these proteins are suitable for generating novel antibodies against the F8 domain and F8 domain-capturing affinity columns by enabling their conjugation to GST-capturing beads. Additionally, the recombinant F8 domains produced herein can be used for various studies, which include investigating the explicit roles of the F8 domain in the coagulation process, with domain-specific binding partners, and antibodies.

Calculation of π and Classification of Self-avoiding Lattices via DNA Configuration.

Numerical simulation (e.g. Monte Carlo simulation) is an efficient computational algorithm establishing an integral part in science to understand complex physical and biological phenomena related with stochastic problems. Aside from the typical numerical simulation applications, studies calculating numerical constants in mathematics, and estimation of growth behavior via a non-conventional self-assembly in connection with DNA nanotechnology, open a novel perspective to DNA related to computational physics. Here, a method to calculate the numerical value of π, and way to evaluate possible paths of self-avoiding walk with the aid of Monte Carlo simulation, are addressed. Additionally, experimentally obtained variation of the π as functions of DNA concentration and the total number of trials, and the behaviour of self-avoiding random DNA lattice growth evaluated through number of growth steps, are discussed. From observing experimental calculations of π (πexp) obtained by double crossover DNA lattices and DNA rings, fluctuation of πexp tends to decrease as either DNA concentration or the number of trials increases. Based upon experimental data of self-avoiding random lattices grown by the three-point star DNA motifs, various lattice configurations are examined and analyzed. This new kind of study inculcates a novel perspective for DNA nanostructures related to computational physics and provides clues to solve analytically intractable problems.

Growth and Detachment of 5 Helix DNA Ribbons.

We report on the concentration-dependent surface-assisted growth and time-temperature-dependent detachment of one-dimensional 5 helix DNA ribbons (5HR) on a mica substrate. The growth coverage ratio was determined by varying the concentration of the 5HR strands in a test tube, and the detachment rate of 5HR on mica was determined by varying the incubation time at a fixed temperature on a heat block. The topological changes in the concentration-dependent attachment and the time-temperature-dependent detachment for 5HR on mica were observed via atomic force microscopy. The observations indicate that 5HR started to grow on mica at ~10 nM and provided full coverage at ~50 nM. In contrast, 5HR at 65 °C started to detach from mica after 5 min and was completely removed after 10 min. The growth and detachment coverage show a sinusoidal variation in the growth ratio and a linear variation with a rate of detachment of 20%/min, respectively. The physical parameters that control the stability of the DNA structures on a given substrate should be studied to successfully integrate DNA structures for physical and chemical applications.