Citation:
TIAN Zhenhua, ZHANG Hao, YU Yuanhua. Turbidimetry Assay for Detection of beta-Glucan by Gαa Binding Protein[J]. Chinese Journal of Applied Chemistry,
;2016, 33(6): 727-732.
doi:
10.11944/j.issn.1000-0518.2016.06.150325
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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 KD 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.
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