Abstract:

In this paper, a beta-glucan binding protein of horseshoe crab(limulus polyphemus) factor G-subunit α fragment-a was cloned, expressed and purified. A turbidimetry assay for the detection of beta-glucan was established. The DNA fragment of the binding protein gene was cloned into an expressional plasmid pET-15b. The protein was expressed with 6x-Histag on N-terminal and purified with Ni-column. The purified protein has relative molecular mass of 40000(1251 bp) on SDS-PAGE and the concentration is 2 g/L with the purity of >98%. The highly purified protein can combine with beta-glucan specifically with an affinity constant K_D of 3.14×10^-9 mol/L. The absorbance measurement at 340 nm using the ultraviolet spectrophotometer shows that the absorbance is linearly correlated with the concentration of beta-glucan. The linear range is 3.125~200 mg/L, R² is 0.997, and the detection limit is 3.125 mg/L. The novel turbidimetry assay for the detection of beta-glucan has low cost, rapid, and highly sensitive and specific.

Key words: horseshoe crab factor G-subunit αfragment-a binding protein, beta-glucan, turbidimetry assay